Method for transposase-mediated spatial tagging and analyzing genomic dna in a biological sample

ABSTRACT

The present disclosure relates to materials and methods for spatially analyzing nucleic acids that have been fragmented with a transposase enzyme, alone or in combination with other types of analytes.

CROSS-REFERENCE TO RELATED APPLICATIONS

Pursuant to 35 U.S.C. § 120, this application is a continuation of U.S.Pat. 17/668,241, filed Mar. 7, 2022 which is a continuation of U.S. Pat.Application Serial No. 16/876,682, filed May 18, 2020, which is acontinuation of International Patent Application PCT/US2019/048425, withan international filing date of Aug. 27, 2019, which claims priority toU.S. Provisional Pat. Application No. 62/724,483, filed Aug. 29, 2018,U.S. Provisional Pat. Application No. 62/779,342, U.S. Provisional Pat.Application No. 62/723,970, filed Aug. 28, 2018, U.S. Provisional Pat.Application No. 62/723,972, filed Aug. 28, 2018, U.S. Provisional Pat.Application No. 62/724,487, filed Aug. 29, 2018, U.S. Provisional Pat.Application No. 62/724,489, filed Aug. 29, 2018, U.S. Provisional Pat.Application No. 62/788,905, filed Jan. 6, 2019, U.S. Provisional Pat.Application No. 62/788,871, filed Jan. 6, 2019, U.S. Provisional Pat.Application No. 62/788,897, filed Jan. 6, 2019, U.S. Provisional Pat.Application No. 62/788,885, filed Jan. 6, 2019, U.S. Provisional Pat.Application No. 62/822,565, filed Mar. 22, 2019, U.S. Provisional Pat.Application No. 62/822,592, filed Mar. 22, 2019, U.S. Provisional Pat.Application No. 62/822,627, filed Mar. 22, 2019, U.S. Provisional Pat.Application No. 62/822,605, filed Mar. 22, 2019, U.S. Provisional Pat.Application No. 62/812,219, filed Feb. 28, 2019, U.S. Provisional Pat.Application No. 62/839,223, filed Apr. 26, 2019 and U.S. ProvisionalPat. Application No. 62/858,331, filed on Jun. 7, 2019. The contents ofeach of these applications are incorporated herein by reference in theirentireties.

SEQUENCE LISTING

This application contains a Sequence Listing that has been submittedelectronically as an XML file named “47706-0036005.XML.” The XML file,created on Sep. 19, 2022, is 35,726 bytes in size. The material in theXML file is hereby incorporated by reference in its entirety.

BACKGROUND

Cells within a tissue of a subject have differences in cell morphologyand/or function due to varied analyte levels (e.g., gene and/or proteinexpression) within the different cells. The specific position of a cellwithin a tissue (e.g., the cell’s position relative to neighboring cellsor the cell’s position relative to the tissue microenvironment) canaffect, e.g., the cell’s morphology, differentiation, fate, viability,proliferation, behavior, and signaling and cross-talk with other cellsin the tissue.

Spatial heterogeneity has been previously studied using techniques thatonly provide data for a small handful of analytes in the contact of anintact tissue or a portion of a tissue, or provide a lot of analyte datafor single cells, but fail to provide information regarding the positionof the single cell in a parent biological sample (e.g., tissue sample).

Chromatin structure can be different between cells in a biologicalsample or between biological samples from the same tissue. Assayingdifferences in accessible chromatin can be indicative oftranscriptionally active sequences, e.g., genes, in a particular cell.Further understanding the transcriptionally active regions withinchromatin will enable identification of which genes contribute to acell’s function and/or phenotype.

SUMMARY

The present disclosure generally describes methods for spatiallyanalyzing genomic DNA present in a biological sample. In one aspect, themethod comprises providing an array with a plurality of capture probessuch that a capture probe of the plurality comprises a spatial barcodeand a capture domain; permeabilizing the biological sample underconditions sufficient to make the genomic DNA in the biological sampleaccessible to a transposon insertion; providing a transposon sequenceand a transposase enzyme to the biological sample under conditionswherein the transposon sequence is inserted into the genomic DNA;allowing the transposase enzyme to excise the inserted transposonsequence from the genomic DNA thus generating fragmented genomic DNA;contacting the biological sample comprising the fragmented genomic DNAwith an array under conditions such that a capture probe interacts withthe fragmented genomic DNA; and correlating the location of the captureprobe on the array to a location in the biological sample, therebyspatially analyzing the fragmented genomic DNA.

In some embodiments, the array comprising a plurality of capture probesare provided on a substrate. In some embodiments, the array comprisingthe plurality of capture probes is provided on a feature. In someembodiments, the capture probe is directly or indirectly attached. Insome embodiments, the array comprising the plurality of capture probesis provided on the feature on the substrate. In some embodiments, thesubstrate comprises a microfluidic channel. In some embodiments, thecapture probe further comprises one or more of a cleavage domain, afunctional domain, and a unique identifier, or combinations thereof.

In some embodiments, a further migration step comprising a step whereinthe fragmented genomic DNA is migrated to the substrate. In someembodiments, the migration step is an active migration step comprisingapplying an electric field to the fragmented genomic DNA. In someembodiments, the migration step is a passive migration step comprisingdiffusion. In some embodiments, the migration of the fragmented genomicDNA from the biological sample comprises exposing the biological sampleand the feature to heat. In some embodiments, the biological sample isimmobilized on the substrate.

In some embodiments, the transposase enzyme is a dimer comprised of afirst monomer complexed with a first adapter comprising a transposon endsequence and a sequence complementary to the capture domain and whereina second monomer is complexed with a second adapter comprising atransposon end sequence and a second adapter sequence, wherein thetransposase enzyme ligates the first adapter and the second adapter tothe fragmented genomic DNA. In some embodiments, the first adapter andthe second adapter have a 5′ end and a 3′ end, wherein the 5′ end isphosphorylated in situ. In some embodiments, prior to fragmenting theDNA, the 5′ end of the first adapter complexed with the first monomerand the second adapter complexed with the second monomer arephosphorylated. In some embodiments, the step of phosphorylating the 5′end of the first adapter complexed with the first monomer and the secondadapter complexed with the second monomer comprises contacting a firstmonomer:first adapter complex and a second monomer: second adaptercomplex with a polynucleotide kinase in the presence of ATP.

In some embodiments, the capture domain of the capture probe comprises asequence that hybridizes to the sequence complementary to the capturedomain of the first adapter. In some embodiments, the capture probe is apartially double stranded molecule comprising a first strand comprisingthe capture domain hybridized to a second strand, and wherein the firststrand templates the ligation of the first adapter to the second strand.In some embodiments, the first adapter sequence complementary to thecapture domain, or portion thereof, hybridized to the capture probetemplates the ligation and ligating the 5′ end of the first adapter tothe 3′ end of the capture probe. In some embodiments, the capture probecomprises a surface probe and a splint oligonucleotide and the splintoligonucleotide comprises a sequence complementary to a hybridizationdomain of the surface probe. In some embodiments, the splintoligonucleotide comprises the capture domain with a sequencecomplementary to the first adapter, or portion thereof. In someembodiments, the splint oligonucleotide hybridizes to the first adapter,or portion thereof, and to the hybridization domain of the surfaceprobe, or portion thereof. In some embodiments, ligation is performed inthe presence of the splint oligonucleotide, thereby ligating the surfaceprobe of the capture probe and the first adapter.

In some embodiments, the fragmented genomic DNA hybridized to thecapture probe by the first adapter is an extension template used toproduce an extended capture probe that comprises the sequences of thespatial barcode and a sequence complementary to the fragmented genomicDNA. In some embodiments, the capture probe hybridized to the fragmentedgenomic DNA is extended with a DNA polymerase. In some embodiments, theDNA polymerase has strand displacement activity. In some embodiments, afurther step of gap repair of single stranded breaks in the fragmentedgenomic DNA.

In some embodiments, the sequence complementary to the capture domain isa unique sequence. In some embodiments, the capture probe is ligated tothe fragmented genomic DNA by a DNA ligase enzyme. In some embodiments,the transposase enzyme is a Tn5 transposase, or a functional derivativethereof. In some embodiments, the Tn5 transposase enzyme comprises asequence having at least 80% identity to SEQ ID NO: 1. In someembodiments, the transposase enzyme is a Mu transposase, or thefunctional derivative thereof. In some embodiments, the Mu transposaseenzyme comprises a sequence having at least 80% identity to SEQ ID NO:2. In some embodiments, the transposon end sequence comprises a sequencehaving at least 80% identity to SEQ ID NO. 8. In some embodiments, thetransposon end sequence comprises a sequence having at least 80%identity to any one of SEQ ID NO: 9 to 14.

In some embodiments, permeabilizing the biological sample is performedunder a chemical permeabilization condition, an enzymaticpermeabilization condition, or both. In some embodiments, the chemicalpermeabilization condition comprises contacting the biological samplewith an alkaline solution. In some embodiments, the enzymaticpermeabilization condition comprises contacting the biological samplewith an acidic solution comprising a protease enzyme. In someembodiments, the protease enzyme is an aspartyl protease, preferably apepsin enzyme, a pepsin-like enzyme, or the functional equivalentthereof. In some embodiments, the pepsin enzyme, the pepsin-like enzyme,or the functional equivalent thereof, comprises a sequence having atleast 80% identity to SEQ ID NO: 3 or 4.

In some embodiments, the enzymatic permeabilization condition comprisescontacting the biological sample with a zinc endopeptidase, acollagenase enzyme, a collagenase-like enzyme, or a functionalequivalent thereof; a serine protease, a proteinase K enzyme, aproteinase K-like enzyme, or a functional equivalent thereof; or both.In some embodiments, the collagenase enzyme, the collagenase-likeenzyme, or the functional equivalent thereof comprises a sequence havingat least 80% identity to SEQ ID NO: 5 or 6. In some embodiments, theproteinase K enzyme, the proteinase K-like enzyme, or the functionalequivalent thereof comprises a sequence having at least 80% identity toSEQ ID NO: 7.

In some embodiments, the fragmented genomic DNA hybridized to thecapture probe as the extension template generates a DNA molecule. Insome embodiments, the fragmented genomic DNA hybridized to the captureprobe acts as a ligation template to generate a DNA molecule. In someembodiments, the step comprising a step of analyzing the generated DNAmolecule. In some embodiments, the step of analyzing the DNA moleculeincludes sequencing. In some embodiments, the step of correlating thespatial barcode of the capture probe with the fragmented genomic DNAassociated with the capture probe spatially analyzes the fragmentedgenomic DNA. In some embodiments, the biological sample is imaged beforeor after contacting the biological sample with the substrate.

In a another aspect, the present disclosure generally describes a kitfor use in a method of spatially detecting nucleic acids of a biologicalsample, wherein the kit comprises any two or more of an array on whichplurality of capture probes are present; one or more biological samplepermeabilization reagents; one or more transposase enzymes; one or morereverse transcriptases; and one or more cleavage enzymes.

In a different aspect, the present disclosure generally describes amethod for spatial analysis of genomic DNA and RNA present in abiological sample wherein an array is provided and the array comprises aplurality of capture probes, wherein a first capture probe of theplurality of capture probes comprises a spatial barcode and a firstcapture domain, and wherein a second capture probe of the plurality ofcapture probes comprises the spatial barcode and a second capturedomain; permeabilizing the biological sample under conditions sufficientto make the genomic DNA in the biological sample accessible totransposon insertion; providing a transposon sequence and a transposaseenzyme to the biological sample under conditions wherein the transposonsequence is inserted into the genomic DNA; allowing the transposaseenzyme to excise the inserted transposon sequence from the genomic DNA,thereby generating fragmented genomic DNA; contacting the biologicalsample comprising the fragmented genomic DNA and RNA with the arrayunder conditions where the first capture domain interacts with thefragmented genomic DNA and the second capture domain interacts with theRNA; and correlating the location of the first capture probe on thearray to a location in the biological sample and correlating thelocation of the second capture probe on the array to a location in thebiological sample, thereby spatially analyzing the fragmented genomicDNA and RNA at the location in the biological sample.

In some embodiments, the RNA is a mRNA. In some embodiments, the firstcapture domain and the second capture domain are identical. In someembodiments, the first capture domain and the second capture domaincomprise a homopolymeric poly (T) sequence. In some embodiments, thefirst capture domain and the second capture domain are different. Insome embodiments, the first capture domain comprises a random sequenceand the second capture domain comprises a poly (T) sequence. In someembodiments, the array comprising the plurality of capture probes isprovided on a substrate. In some embodiments, the array comprising theplurality of capture probes is provided on a feature. In someembodiments, the feature comprises the first capture probe, the secondcapture probe, or both. In some embodiments, the first capture probe,the second capture probe, or both, are directly or indirectly attached.In some embodiments, the array comprising the plurality of captureprobes is provided on the feature on the substrate. In some embodiments,the substrate comprises a microfluidic channel. In some embodiments, thefirst capture probe, the second capture probe, or both, comprise one ormore of a cleavage domain, a functional domain, and a unique identifier,or combinations thereof.

In some embodiments, there is a migration step wherein the fragmentedgenomic DNA and the RNA are migrated to the substrate. In someembodiments, the migration step is an active migration step. In someembodiments, the migration step is a passive migration step. In someembodiments, the migration of the fragmented genomic DNA and the RNAfrom the biological sample comprises exposing the biological sample toheat. In some embodiments, the biological sample is immobilized on thesubstrate.

In some embodiments, the fragmented genomic DNA is repaired by ligatingbreaks with a ligase enzyme. In some embodiments, single stranded breaksin the fragmented genomic DNA undergo gap repair. In some embodiments, asequence complementary to the first capture domain of the first captureprobe is introduced to the fragmented genomic DNA. In some embodiments,the first capture domain of the first capture probe hybridizes to thesequence complementary to the capture domain introduced to thefragmented genomic DNA. In some embodiments, the random sequence of thefirst capture domain hybridizes the fragmented genomic DNA. In someembodiments, the second capture domain of the second capture probehybridizes to a complementary sequence in the mRNA. In some embodiments,the sequence complementary to the first capture domain and thecomplementary sequence in the mRNA is a homopolymeric sequence. In someembodiments, the homopolymeric sequence is a poly(A) sequence.

In some embodiments, extension of the first capture probe using thefragmented genomic DNA as an extension template, and extension of thesecond capture probe using the RNA as an extension template isperformed. In some embodiments, extending the first capture probe isperformed with a DNA polymerase. In some embodiments, extending thesecond capture probe is performed with reverse transcriptase.

In some embodiments, transposase is a Tn5 transposase, or a functionalderivative thereof. In some embodiments, the Tn5 transposase enzymecomprises a sequence having at least 80% identity to SEQ ID NO: 1. Insome embodiments, the transposase enzyme is a Mu transposase enzyme, ora functional derivative thereof. In some embodiments, the Mu transposaseenzyme comprises a sequence having at least 80% identity to SEQ ID NO:2. In some embodiments, the transposase enzyme is complexed with anadapter comprising a transposon end sequence. In some embodiments, thetransposon end sequence comprises a sequence having at least 80%identity to SEQ ID NO: 8. In some embodiments, the transposon endsequence comprises a sequence having at least 80% identity to any one ofSEQ ID NO: 9 to 14.

In some embodiments, a step of permeabilizing the biological sample isperformed. In some embodiments, 7. The method of any one of claims 51 to86, wherein permeabilizing the biological sample is performed under achemical permeabilization condition, an enzymatic permeabilizationcondition, or both. In some embodiments, the chemical permeabilizationcondition comprises contacting the biological sample with an alkalinesolution. In some embodiments, the enzymatic permeabilization conditioncomprises contacting the biological sample with an acidic solutioncomprising a protease enzyme. In some embodiments, the protease enzymeis an aspartyl protease, preferably a pepsin enzyme, a pepsin-likeenzyme, or a functional equivalent thereof. In some embodiments, thepepsin enzyme, the pepsin-like enzyme, or functional equivalent thereof,comprises a sequence having at least 80% identity to SEQ ID NO: 3 or 4.In some embodiments, the enzymatic permeabilization condition comprisescontacting the biological sample with a zinc endopeptidase, acollagenase enzyme, a collagenase-like enzyme, or a functionalequivalent thereof; a serine protease, a proteinase K enzyme, aproteinase K-like enzyme, or a functional equivalent thereof; or both.In some embodiments, the collagenase enzyme, the collagenase-likeenzyme, or the functional equivalent thereof comprises a sequence havingat least 80% identity to SEQ ID NO: 5 or 6. I some embodiments, theproteinase K enzyme, the proteinase K-like enzyme, or the functionalequivalent thereof comprises a sequence having at least 80% identity toSEQ ID NO: 7.

In some embodiments, step of analyzing the DNA molecule includessequencing. In some embodiments, correlating the spatial barcode of thefirst capture probe with the fragmented genomic DNA associated with thefirst capture probe spatially analyzes the fragmented genomic DNA. Insome embodiments, correlating the spatial barcode of the second captureprobe with the mRNA associated with the second capture probe spatiallyanalyzes the mRNA. In some embodiments, the biological sample is imagedbefore or after contacting the biological sample with the substrate.

All publications, patents, patent applications, and informationavailable on the internet and mentioned in this specification are hereinincorporated by reference to the same extent as if each individualpublication, patent, patent application, or item of information wasspecifically and individually indicated to be incorporated by reference.To the extent publications, patents, patent applications, and items ofinformation incorporated by reference contradict the disclosurecontained in the specification, the specification is intended tosupersede and/or take precedence over any such contradictory material.

Where values are described in terms of ranges, it should be understoodthat the description includes the disclosure of all possible sub-rangeswithin such ranges, as well as specific numerical values that fallwithin such ranges irrespective of whether a specific numerical value orspecific sub-range is expressly stated.

The term “each,” when used in reference to a collection of items, isintended to identify an individual item in the collection but does notnecessarily refer to every item in the collection, unless expresslystated otherwise, or unless the context of the usage clearly indicatesotherwise.

Various embodiments of the features of this disclosure are describedherein. However, it should be understood that such embodiments areprovided merely by way of example, and numerous variations, changes, andsubstitutions can occur to those skilled in the art without departingfrom the scope of this disclosure. It should also be understood thatvarious alternatives to the specific embodiments described herein arealso within the scope of this disclosure.

DESCRIPTION OF DRAWINGS

The following drawings illustrate certain embodiments of the featuresand advantages of this disclosure. These embodiments are not intended tolimit the scope of the appended claims in any manner. Like referencesymbols in the drawings indicate like elements.

FIG. 1 shows an exemplary spatial analysis workflow.

FIG. 2 shows an exemplary spatial analysis workflow.

FIG. 3 shows an exemplary spatial analysis workflow.

FIG. 4 shows an exemplary spatial analysis workflow.

FIG. 5 shows an exemplary spatial analysis workflow.

FIG. 6 is a schematic diagram showing an example of a barcoded captureprobe, as described herein.

FIG. 7 is a schematic illustrating a cleavable capture probe, whereinthe cleaved capture probe can enter into a non-permeabilized cell andbind to target analytes within the sample.

FIG. 8 is a schematic diagram of an exemplary multiplexedspatially-labelled feature.

FIG. 9 is a schematic diagram of an exemplary analyte capture agent.

FIG. 10 is a schematic diagram depicting an exemplary interactionbetween a feature-immobilized capture probe 1024 and an analyte captureagent 1026.

FIGS. 11A, 11B, and 11C are schematics illustrating how streptavidincell tags can be utilized in an array-based system to produce aspatially-barcoded cells or cellular contents.

FIG. 12 is a schematic showing the arrangement of barcoded featureswithin an array.

FIG. 13 is a schematic illustrating a side view of a diffusion-resistantmedium, e.g., a lid.

FIGS. 14A and 14B are schematics illustrating expanded FIG. 14A and sideviews FIG. 14B of an electrophoretic transfer system configured todirect transcript analytes toward a spatially-barcoded capture probearray.

FIGS. 15A-G is a schematic illustrating an exemplary workflow protocolutilizing an electrophoretic transfer system.

FIG. 16 shows an example of a microfluidic channel structure 1600 forpartitioning dissociated sample (e.g. biological particles or individualcells from a sample).

FIG. 17A shows an example of a microfluidic channel structure 1700 fordelivering spatial barcode carrying beads to droplets.

FIG. 17B shows a cross-section view of another example of a microfluidicchannel structure 1750 with a geometric feature for controlledpartitioning.

FIG. 17C shows a workflow schematic.

FIG. 18 is a schematic depicting cell tagging using either covalentconjugation of the analyte binding moiety to the cell surface ornon-covalent interactions with cell membrane elements.

FIG. 19 is a schematic depicting cell tagging using eithercell-penetrating peptides or delivery systems.

FIG. 20A is a workflow schematic illustrating exemplary, non-limiting,non-exhaustive steps for “pixelating” a sample, wherein the sample iscut, stamped, microdissected, or transferred by hollow-needle ormicroneedle, moving a small portion of the sample into an individualpartition or well.

FIG. 20B is a schematic depicting multi-needle pixilation, wherein anarray of needles punched through a sample on a scaffold and intonanowells containing gel beads and reagents below. Once the needle is inthe nanowell, the cell(s) are ejected.

FIG. 21 shows a workflow schematic illustrating exemplary, non-limiting,non-exhaustive steps for dissociating a spatially-barcoded sample foranalysis via droplet or flow cell analysis methods.

FIGS. 22A-D is a schematic diagram showing an example of spatiallyprocessing DNA from a biological sample.

FIGS. 23A-C is a schematic diagram showing an example of a spatialATAC-seq method.

FIGS. 24A-C is a schematic diagram showing an example of multiplexdetection of analytes in a biological sample.

FIG. 25 is a schematic diagram showing a representative workflow of theinvention.

FIG. 26 is a schematic diagram showing a representative workflow of theprocedure used to investigate Tn5 transposase/transposome efficiency.

FIG. 27 is a schematic diagram showing a representative workflow of theprocedure used to investigate tagmentation conditions in immobilizedtissue sections.

FIG. 28 is a schematic diagram showing a representative workflow of theprocedure used to investigate hybridization and ligation conditions ofphosphorylated DNA tagments.

FIG. 29 shows DNA fragment analysis of a reference tagmentation reactionperformed in a cellular suspension as described (Corces, M.R., et. al.,Lineage-specific and single-cell chromatin accessibility charts humanhematopoiesis and leukemia evolution, Nat Genetic. vol. 48(10): pp.1193-1203 (2016)). Fragment distribution analysis is used to determinethe success of open chromatin tagmentation, wherein a successfultagmentation reaction of accessible chromatin reveals a periodicity(approx. 170-180 bp; nucleosome-wrapped DNA and PCR handles) in the sizeof PCR-amplified nucleosome-protected DNA fragments.

FIGS. 30A-E shows a DNA fragment analysis of tagmentation reactionsperformed according to the workflow in FIG. 27 comparing differentdetergents in the permeabilization step performed for 10 minutes at 25°C.: FIG. 30A) no detergent; FIG. 30B) 0.1% Triton-X-100; FIG. 30C)IGEPAL 0.1%; FIG. 30D) Tween 0.1%, Digitonin 0.01% and NP-40 0.1%. InFIG. 30E), insert size distribution analysis on a tissue sectionpermeabilized with IGEBAL 0.1% and processed as in (Chen 2016 Nat Meth)fails to reveal a prominent nucleosome periodicity.

FIGS. 31A-D shows a DNA fragment analysis of tagmentation reactionsperformed according to the workflow in FIG. 27 comparing differentprotease treatments (3 minutes) on an immobilized tissue section: FIG.31A) Pepsin (0.1 mg/ml) in presence of 100 mM HCL; FIG. 31B) Pepsin (0.5mg/ml) in the presence of 0.5 M acetic acid; FIG. 31C) Pepsin (0.1mg/ml) in the presence of 0.5 M acetic acid; and FIG. 31D) Proteinase K.

FIGS. 32A-C shows a DNA fragment analysis of tagmentation reactionsperformed according to the workflow in FIG. 27 comparing differentpermeabilization treatments on an immobilized tissue section: FIG. 32A)Pepsin (0.1 mg/ml) in the presence of 0.5 acetic acid; FIG. 32B)chemical permeabilization using 1X Exonuclease-I buffer (67 mMGlycine-KOH, 6.7 mM MgCl₂, 10 mM β-ME); and FIG. 32C) Collagenase.

FIGS. 33A-C shows a DNA fragment analysis of tagmentation reactionsperformed according to the workflow in FIG. 27 comparing different Tn5assembly methods on an immobilized tissue section: FIG. 33A) MEDS-Tn5assembled on column as in (Picelli, S., et. al., Tn5 transposase andtagmentation procedures for massively scaled sequencing projects; GenomeRes., vol. 24, 2033-2040 (2014)); FIG. 33B) MEDS-Tn5 assembled insolution as in (Picelli et al., 2014, supra); FIG. 33C) MEDS-Tn5assembly with 5′ phosphorylated oligonucleotides assembled in solution.

FIG. 34 is a schematic diagram showing a representation of the tests toassess the effect of post-assembly T4-PNK phosphorylation and reactionconditions on MEDS Tn5 complexes.

FIGS. 35A-D shows a DNA fragment analysis of tagmentation reactionsperformed according the workflow in FIG. 26 investigating thecompatibility of post-assembly 5′ phosphorylation with DNA tagmentationFIG. 35A) on-column assembled MEDS-AB-Tn5 as in (Picelli et al., 2014,supra): FIG. 35B) as FIG. 35A) but exposed to T4-PNK reaction conditionsfor 30 min at 37° C.; FIG. 35C) as FIG. 35B) but including T4-PNKenzyme; and FIG. 35D) a bar chart showing the quantification of therelative proportions of nucleosome-protected fragments recovered inFIGS. 35A-C.

FIGS. 36A-B shows photographs of arrays generated according to theworkflow in FIG. 28 , depicting the ligation efficiency of DNA tagmentsonto capture probe oligonucleotides (FIG. 36A) without and (FIG. 36B)with post-assembly phosphorylation.

FIG. 37 is a schematic depicting a representative embodiment of theinvention in which tagments are gap-filled with a polymerase withslippery activity (e.g., stuttering), creating poly-A-sticky end (3′overhang) at the 3′-ends (mimicking an mRNA poly(A)-tail) with aterminal transferase and subsequent hybridization to the capture domainof a capture probe (this embodiment would allow simultaneoushybridization of mRNA-transcripts). Alternatively, a polymerase can beused to extend the tagment prior to capture.

FIG. 38 is a schematic diagram of a representative embodiment of theinvention in which tagments are ligated to partially double strandedcapture probes using the capture domain strand of the capture probe(e.g., a capture domain oligonucleotide) as a ligation template.

FIG. 39 is a schematic diagram showing a representative workflow of theprocedure used to investigate ligation of phosphorylated DNA tagmentsfrom a whole human genome and downstream qPCR analysis.

FIG. 40 shows a schematic representation of an exemplary oligonucleotidecapture strategy and the respective sequences. Readout is performed byqPCR with oligonucleotides specific to tagments successfully ligated tothe surface (e.g., A-short and Nextera reverse) or to all tagments(e.g., Nextera forward and Nextera reverse).

FIG. 41A is a schematic diagram of a substrate outline under variousexperimental conditions following the workflow shown in FIG. 39(ligation of phosphorylated DNA fragments from a whole human genome).

FIG. 41B shows a DNA fragment analysis of tagmentation reactionsperformed according to the workflow shown in FIG. 39 . The PCR primerpair “Ashort-Next” covers both the surface probe and the tagment. Thisprimer pair only results in a PCR product when hybridization andligation have occurred. Samples 1 and 2 represent tagments withphosphate groups added to facilitate ligation. Samples 3 and 4 hadtagments lacking phosphate groups and served as negative controls andsamples 5 and 6 had MQ water instead of tagments. Further, a pair ofNextera primers (“NEXT ONLY”, samples 7-11) show the PCR products whenboth ligation and hybridization have occurred, thus resulting in asignal from the D and E wells.

FIG. 41C shows a graph showing an alignment of PCR products. The graphshows ligation (ligated qPCR products) with “Ashort-Next” primers,whereas minimal ligation occurred in all four negative controls.

FIG. 42 shows a schematic diagram showing a representative workflow ofthe procedure used to investigate permeabilization and tagmentationconditions of DNA tagments in immobilized tissue sections. Results frompartial protein digestion with trypsin or Proteinase-K duringpre-permeabilization are shown.

FIGS. 43A-C shows graphs showing the effect of collagenase treatmentfollowed by either Proteinase-K (FIG. 43A) or trypsin (FIG. 43B)pre-permeabilization on tagmentation efficiency according to theworkflow shown in FIG. 42 . The experiment was performed in duplicate.Proteinase-K pre-permeabilization treatment resulted in uniformly highsignal of amplified tagments compared to trypsin pre-permeabilizationtreatment or (FIG. 43C) the negative control (phosphate negativetagments).

FIG. 44 shows a schematic diagram showing a representative workflow ofthe procedure used to investigate the capture of DNA tagments fromimmobilized tissue sections.

FIGS. 45A-D shows graphs and photographs showing the successful captureof DNA tagments from immobilized tissue sections according to theworkflow shown in FIG. 44 with collagenase and Proteinase-Kpre-permeabilization treatment. Each experiment was performed induplicate: one experiment for PCR downstream analysis and one experimentfor hybridization using a fluorescently labeled (Cy5) oligonucleotidecomplementary to the ligated tagments. The phosphate positive samplesresulted in detectable signal (FIG. 45A and FIG. 45B), whereas thephosphate negative sample did not (FIG. 45C). FIG. 45D shows ahematoxylin-eosin image (left) and the corresponding spatial pattern ofligated DNA tagments (right) showing successful DNA capture from thetissue section.

FIG. 46A is a schematic diagram showing an example sample handlingapparatus that can be used to implement various steps and methodsdescribed herein.

FIG. 46B is a schematic diagram showing an example imaging apparatusthat can be used to obtain images of biological samples, analytes, andarrays of features.

FIG. 46C is a schematic diagram of an example of a control unit of theapparatus of FIGS. 46A and 46B.

DETAILED DESCRIPTION I. Introduction

This disclosure describes apparatus, systems, methods, and compositionsfor spatial analysis of biological samples. This section in particulardescribes certain general terminology, analytes, sample types, andpreparative steps that are referred to in later sections of thedisclosure.

(A) Spatial Analysis

Tissues and cells can be obtained from any source. For example, tissuesand cells can be obtained from single-cell or multicellular organisms(e.g., a mammal). Tissues and cells obtained from a mammal, e.g., ahuman, often have varied analyte levels (e.g., gene and/or proteinexpression) which can result in differences in cell morphology and/orfunction. The position of a cell within a tissue can affect, e.g., thecell’s fate, behavior, morphology, and signaling and cross-talk withother cells in the tissue. Information regarding the differences inanalyte levels (gene and/or protein expression) within different cellsin a tissue of a mammal can also help physicians select or administer atreatment that will be effective in the single-cell or multicellularorganisms (e.g., a mammal) based on the detected differences in analytelevels within different cells in the tissue. Differences in analytelevels within different cells in a tissue of a mammal can also provideinformation on how tissues (e.g., healthy and diseased tissues) functionand/or develop. Differences in analyte levels within different cells ina tissue of a mammal can also provide information of differentmechanisms of disease pathogenesis in a tissue and mechanism of actionof a therapeutic treatment within a tissue. Differences in analytelevels within different cells in a tissue of a mammal can also provideinformation on drug resistance mechanisms and the development of thesame in a tissue of a mammal. Differences in the presence or absence ofanalytes within different cells in a tissue of a multicellular organism(e.g., a mammal) can provide information on drug resistance mechanismsand the development of the same in a tissue of a multicellular organism.

The spatial analysis methodologies provide for the detection ofdifferences in an analyte level (e.g., gene and/or protein expression)within different cells in a tissue of a mammal or within a single cellfrom a mammal. For example, spatial analysis methodologies can be usedto detect the differences in analyte levels (e.g., gene and/or proteinexpression) within different cells in histological slide samples, thedata from which can be reassembled to generate a three-dimensional mapof analyte levels (e.g., gene and/or protein expression) of a tissuesample obtained from a mammal, e.g., with a degree of spatial resolution(e.g., single-cell resolution).

Spatial heterogeneity in developing systems has typically been studiedvia RNA hybridization, immunohistochemistry, fluorescent reporters, orpurification or induction of pre-defined subpopulations and subsequentgenomic profiling (e.g., RNA-seq). Such approaches, however, rely on arelatively small set of pre-defined markers, therefore introducingselection bias that limits discovery. These prior approaches also relyon a priori knowledge. Spatial RNA assays traditionally relied onstaining for a limited number of RNA species. In contrast, single-cellRNA-sequencing allows for deep profiling of cellular gene expression(including non-coding RNA), but the established methods separate cellsfrom their native spatial context.

Current spatial analysis methodologies provide a vast amount of analytelevel and/or expression data for a variety of multiple analytes within asample at high spatial resolution, e.g., while retaining the nativespatial context. Spatial analysis methods include, e.g., the use of acapture probe including a spatial barcode (e.g., a nucleic acid sequencethat provides information as to the position of the capture probe withina cell or a tissue sample (e.g., mammalian cell or a mammalian tissuesample) and a capture domain that is capable of binding to an analyte(e.g., a protein and/or nucleic acid) produced by and/or present in acell. As described herein, the spatial barcode can be a nucleic acidthat has a unique sequence, a unique fluorophore or a unique combinationof fluorophores, a unique amino acid sequence, a unique heavy metal or aunique combination of heavy metals, or any other unique detectableagent. The capture domain can be any agent that is capable of binding toan analyte produced by and/or present in a cell (e.g., a nucleic acidthat is capable of hybridizing to a nucleic acid from a cell (e.g., anmRNA, genomic DNA, mitochondrial DNA, or miRNA), a substrate or bindingpartner of an analyte, or an antibody that binds specifically to ananalyte). A capture probe can also include a nucleic acid sequence thatis complementary to a sequence of a universal forward and/or universalreverse primer. A capture probe can also include a cleavage site (e.g.,a cleavage recognition site of a restriction endonuclease), aphotolabile bond, a thermosensitive bond, or a chemical-sensitive bond.

The binding of an analyte to a capture probe can be detected using anumber of different methods, e.g., nucleic acid sequencing, fluorophoredetection, nucleic acid amplification, detection of nucleic acidligation, and/or detection of nucleic acid cleavage products. In someexamples, the detection is used to associate a specific spatial barcodewith a specific analyte produced by and/or present in a cell (e.g., amammalian cell).

Capture probes can be, e.g., attached to a surface, e.g., a solid array,a bead, or a coverslip. In some examples, capture probes are notattached to a surface. In some examples, capture probes can beencapsulated within, embedded within, or layered on a surface of apermeable composition (e.g., any of the substrates described herein).For example, capture probes can be encapsulated or disposed within apermeable bead (e.g., a gel bead). In some examples, capture probes canbe encapsulated within, embedded within, or layered on a surface of asubstrate (e.g., any of the exemplary substrates described herein, suchas a hydrogel or a porous membrane).

In some examples, a cell or a tissue sample including a cell arecontacted with capture probes attached to a substrate (e.g., a surfaceof a substrate), and the cell or tissue sample is permeabilized to allowanalytes to be released from the cell and bind to the capture probesattached to the substrate. In some examples, analytes released from acell can be actively directed to the capture probes attached to asubstrate using a variety of methods, e.g., electrophoresis, chemicalgradient, pressure gradient, fluid flow, or magnetic field.

In other examples, a capture probe can be directed to interact with acell or a tissue sample using a variety of methods, e.g., inclusion of alipid anchoring agent in the capture probe, inclusion of an agent thatbinds specifically to, or forms a covalent bond with a membrane proteinin the capture probe, fluid flow, pressure gradient, chemical gradient,or magnetic field.

Non-limiting aspects of spatial analysis methodologies are described inWO 2011/127099, WO 2014/210233, WO 2014/210225, WO 2016/162309, WO2018/091676, WO 2012/140224, WO 2014/060483, U.S. Pat. 10,002,316, U.S.Pat. 9,727,810, U.S. Pat. Application Publication No. 2017/0016053,Rodriques et al., Science 363(6434):1463-1467, 2019; WO 2018/045186, Leeet al., Nat. Protoc. 10(3):442-458, 2015; WO 2016/007839, WO2018/045181, WO 2014/163886, Trejo et al., PLoS ONE 14(2):e0212031,2019, U.S. Pat. Application Publication No. 2018/0245142, Chen et al.,Science 348(6233):aaa6090, 2015, Gao et al., BMC Biol. 15:50, 2017, WO2017/144338, WO 2018/107054, WO 2017/222453, WO 2019/068880, WO2011/094669, U.S. Pat. 7,709,198, U.S. Pat. 8,604,182, U.S. Pat.8,951,726, U.S. Pat. 9,783,841, U.S. Pat. 10,041,949, WO 2016/057552, WO2017/147483, WO 2018/022809, WO 2016/166128, WO 2017/027367, WO2017/027368, WO 2018/136856, WO 2019/075091, U.S. Pat. 10,059,990, WO2018/057999, WO 2015/161173, and Gupta et al., Nature Biotechnol.36:1197-1202, 2018, and can be used herein in any combination. Furthernon-limiting aspects of spatial analysis methodologies are describedherein.

(B) General Terminology

Specific terminology is used throughout this disclosure to explainvarious aspects of the apparatus, systems, methods, and compositionsthat are described. This sub-section includes explanations of certainterms that appear in later sections of the disclosure. To the extentthat the descriptions in this section are in apparent conflict withusage in other sections of this disclosure, the definitions in thissection will control.

(I) Barcode

A “barcode” is a label, or identifier, that conveys or is capable ofconveying information (e.g., information about an analyte in a sample, abead, and/or a capture probe). A barcode can be part of an analyte, orindependent of an analyte. A barcode can be attached to an analyte. Aparticular barcode can be unique relative to other barcodes.

Barcodes can have a variety of different formats. For example, barcodescan include polynucleotide barcodes, random nucleic acid and/or aminoacid sequences, and synthetic nucleic acid and/or amino acid sequences.A barcode can be attached to an analyte or to another moiety orstructure in a reversible or irreversible manner. A barcode can be addedto, for example, a fragment of a deoxyribonucleic acid (DNA) orribonucleic acid (RNA) sample before or during sequencing of the sample.Barcodes can allow for identification and/or quantification ofindividual sequencing-reads (e.g., a barcode can be or can include aunique molecular identifier or “UMI”).

Barcodes can spatially-resolve molecular components found in biologicalsamples, for example, at single-cell resolution (e.g., a barcode can beor can include a “spatial barcode”). In some embodiments, a barcodeincludes both a UMI and a spatial barcode. In some embodiments, abarcode includes two or more sub-barcodes that together function as asingle barcode. For example, a polynucleotide barcode can include two ormore polynucleotide sequences (e.g., sub-barcodes) that are separated byone or more non-barcode sequences.

(II) Nucleic Acid and Nucleotide

The terms “nucleic acid” and “nucleotide” are intended to be consistentwith their use in the art and to include naturally-occurring species orfunctional analogs thereof. Particularly useful functional analogs ofnucleic acids are capable of hybridizing to a nucleic acid in asequence-specific fashion (e.g., capable of hybridizing to two nucleicacids such that ligation can occur between the two hybridized nucleicacids) or are capable of being used as a template for replication of aparticular nucleotide sequence. Naturally-occurring nucleic acidsgenerally have a backbone containing phosphodiester bonds. An analogstructure can have an alternate backbone linkage including any of avariety of those known in the art. Naturally-occurring nucleic acidsgenerally have a deoxyribose sugar (e.g., found in deoxyribonucleic acid(DNA)) or a ribose sugar (e.g. found in ribonucleic acid (RNA)).

A nucleic acid can contain nucleotides having any of a variety ofanalogs of these sugar moieties that are known in the art. A nucleicacid can include native or non-native nucleotides. In this regard, anative deoxyribonucleic acid can have one or more bases selected fromthe group consisting of adenine (A), thymine (T), cytosine (C), orguanine (G), and a ribonucleic acid can have one or more bases selectedfrom the group consisting of uracil (U), adenine (A), cytosine (C), orguanine (G). Useful non-native bases that can be included in a nucleicacid or nucleotide are known in the art.

(III) Probe and Target

A “probe” or a “target,” when used in reference to a nucleic acid orsequence of a nucleic acids, is intended as a semantic identifier forthe nucleic acid or sequence in the context of a method or composition,and does not limit the structure or function of the nucleic acid orsequence beyond what is expressly indicated.

(IV) Oligonucleotide and Polynucleotide

The terms “oligonucleotide” and “polynucleotide” are usedinterchangeably to refer to a single-stranded multimer of nucleotidesfrom about 2 to about 500 nucleotides in length. Oligonucleotides can besynthetic, made enzymatically (e.g., via polymerization), or using a“split-pool” method. Oligonucleotides can include ribonucleotidemonomers (i.e., can be oligoribonucleotides) and/or deoxyribonucleotidemonomers (i.e., oligodeoxyribonucleotides). In some examples,oligonucleotides can include a combination of both deoxyribonucleotidemonomers and ribonucleotide monomers in the oligonucleotide (e.g.,random or ordered combination of deoxyribonucleotide monomers andribonucleotide monomers). An oligonucleotide can be 4 to 10, 10 to 20,21 to 30, 31 to 40, 41 to 50, 51 to 60, 61 to 70, 71 to 80, 80 to 100,100 to 150, 150 to 200, 200 to 250, 250 to 300, 300 to 350, 350 to 400,or 400-500 nucleotides in length, for example. Oligonucleotides caninclude one or more functional moieties that are attached (e.g.,covalently or non-covalently) to the multimer structure. For example, anoligonucleotide can include one or more detectable labels (e.g., aradioisotope or fluorophore).

(V) Subject

A “subject” is an animal, such as a mammal (e.g., human or a non-humansimian), or avian (e.g., bird), or other organism, such as a plant.Examples of subjects include, but are not limited to, a mammal such as arodent, mouse, rat, rabbit, guinea pig, ungulate, horse, sheep, pig,goat, cow, cat, dog, primate (i.e. human or non-human primate); a plantsuch as Arabidopsis thaliana, corn, sorghum, oat, wheat, rice, canola,or soybean; an algae such as Chlamydomonas reinhardtii; a nematode suchas Caenorhabditis elegans; an insect such as Drosophila melanogaster,mosquito, fruit fly, or honey bee; an arachnid such as a spider; a fishsuch as zebrafish; a reptile; an amphibian such as a frog or Xenopuslaevis; a Dictyostelium discoideum; a fungi such as Pneumocystiscarinii, Takifugu rubripes, yeast, Saccharamoyces cerevisiae orSchizosaccharomyces pombe; or a Plasmodium falciparum.

(VI) Genome

A “genome” generally refers to genomic information from a subject, whichcan be, for example, at least a portion of, or the entirety of, thesubject’s gene-encoded hereditary information. A genome can includecoding regions (e.g., that code for proteins) as well as non-codingregions. A genome can include the sequences of some or all of thesubject’s chromosomes. For example, the human genome ordinarily has atotal of 46 chromosomes. The sequences of some or all of these canconstitute the genome.

(VII) Adaptor, Adapter, and Tag

An “adaptor,” an “adapter,” and a “tag” are terms that are usedinterchangeably in this disclosure, and refer to species that can becoupled to a polynucleotide sequence (in a process referred to as“tagging”) using any one of many different techniques including (but notlimited to) ligation, hybridization, and tagmentation. Adaptors can alsobe nucleic acid sequences that add a function, e.g., spacer sequences,primer sequences/sites, barcode sequences, unique molecular identifiersequences.

(VIII) Hybridizing, Hybridize, Annealing, and Anneal

The terms “hybridizing,” “hybridize,” “annealing,” and “anneal” are usedinterchangeably in this disclosure, and refer to the pairing ofsubstantially complementary or complementary nucleic acid sequenceswithin two different molecules. Pairing can be achieved by any processin which a nucleic acid sequence joins with a substantially or fullycomplementary sequence through base pairing to form a hybridizationcomplex. For purposes of hybridization, two nucleic acid sequences are“substantially complementary” if at least 60% (e.g., at least 70%, atleast 80%, or at least 90%) of their individual bases are complementaryto one another.

(IX) Primer

A “primer” is a single-stranded nucleic acid sequence having a 3′ endthat can be used as a substrate for a nucleic acid polymerase in anucleic acid extension reaction. RNA primers are formed of RNAnucleotides, and are used in RNA synthesis, while DNA primers are formedof DNA nucleotides and used in DNA synthesis. Primers can also includeboth RNA nucleotides and DNA nucleotides (e.g., in a random or designedpattern). Primers can also include other natural or syntheticnucleotides described herein that can have additional functionality. Insome examples, DNA primers can be used to prime RNA synthesis and viceversa (e.g., RNA primers can be used to prime DNA synthesis). Primerscan vary in length. For example, primers can be about 6 bases to about120 bases. For example, primers can include up to about 25 bases.

(X) Primer Extension

A “primer extension” refers to any method where two nucleic acidsequences (e.g., a constant region from each of two distinct captureprobes) become linked (e.g., hybridized) by an overlap of theirrespective terminal complementary nucleic acid sequences (i.e., forexample, 3′ termini). Such linking can be followed by nucleic acidextension (e.g., an enzymatic extension) of one, or both termini usingthe other nucleic acid sequence as a template for extension. Enzymaticextension can be performed by an enzyme including, but not limited to, apolymerase and/or a reverse transcriptase.

(XI) Proximity Ligation

A “proximity ligation” is a method of ligating two (or more) nucleicacid sequences that are in proximity with each other through enzymaticmeans (e.g., a ligase). In some embodiments, proximity ligation caninclude a “gap-filling” step that involves incorporation of one or morenucleic acids by a polymerase, based on the nucleic acid sequence of atemplate nucleic acid molecule, spanning a distance between the twonucleic acid molecules of interest (see, e.g., U.S. Pat. 7,264,929, theentire contents of which are incorporated herein by reference).

A wide variety of different methods can be used for proximity ligatingnucleic acid molecules, including (but not limited to) “sticky-end” and“blunt-end” ligations. Additionally, single-stranded ligation can beused to perform proximity ligation on a single-stranded nucleic acidmolecule. Sticky-end proximity ligations involve the hybridization ofcomplementary single-stranded sequences between the two nucleic acidmolecules to be joined, prior to the ligation event itself. Blunt-endproximity ligations generally do not include hybridization ofcomplementary regions from each nucleic acid molecule because bothnucleic acid molecules lack a single-stranded overhang at the site ofligation.

(XII) Nucleic Acid Extension

A “nucleic acid extension” generally involves incorporation of one ormore nucleic acids (e.g., A, G, C, T, U, nucleotide analogs, orderivatives thereof) into a molecule (such as, but not limited to, anucleic acid sequence) in a template-dependent manner, such thatconsecutive nucleic acids are incorporated by an enzyme (such as apolymerase or reverse transcriptase), thereby generating a newlysynthesized nucleic acid molecule. For example, a primer that hybridizesto a complementary nucleic acid sequence can be used to synthesize a newnucleic acid molecule by using the complementary nucleic acid sequenceas a template for nucleic acid synthesis. Similarly, a 3′ polyadenylatedtail of an mRNA transcript that hybridizes to a poly (dT) sequence(e.g., capture domain) can be used as a template for single-strandsynthesis of a corresponding cDNA molecule.

(XIII) PCR Amplification

A “PCR amplification” refers to the use of a polymerase chain reaction(PCR) to generate copies of genetic material, including DNA and RNAsequences. Suitable reagents and conditions for implementing PCR aredescribed, for example, in U.S. Pats 4,683,202, 4,683,195, 4,800,159,4,965,188, and 5,512,462, the entire contents of each of which areincorporated herein by reference. In a typical PCR amplification, thereaction mixture includes the genetic material to be amplified, anenzyme, one or more primers that are employed in a primer extensionreaction, and reagents for the reaction. The oligonucleotide primers areof sufficient length to provide for hybridization to complementarygenetic material under annealing conditions. The length of the primersgenerally depends on the length of the amplification domains, but willtypically be at least 4 bases, at least 5 bases, at least 6 bases, atleast 8 bases, at least 9 bases, at least 10 base pairs (bp), at least11 bp, at least 12 bp, at least 13 bp, at least 14 bp, at least 15 bp,at least 16 bp, at least 17 bp, at least 18 bp, at least 19 bp, at least20 bp, at least 25 bp, at least 30 bp, at least 35 bp, and can be aslong as 40 bp or longer, where the length of the primers will generallyrange from 18 to 50 bp. The genetic material can be contacted with asingle primer or a set of two primers (forward and reverse primers),depending upon whether primer extension, linear or exponentialamplification of the genetic material is desired.

In some embodiments, the PCR amplification process uses a DNA polymeraseenzyme. The DNA polymerase activity can be provided by one or moredistinct DNA polymerase enzymes. In certain embodiments, the DNApolymerase enzyme is from a bacterium, e.g., the DNA polymerase enzymeis a bacterial DNA polymerase enzyme. For instance, the DNA polymerasecan be from a bacterium of the genus Escherichia, Bacillus,Thermophilus, or Pyrococcus.

Suitable examples of DNA polymerases that can be used include, but arenot limited to: E.coli DNA polymerase I, Bsu DNA polymerase, Bst DNApolymerase, Taq DNA polymerase, VENT™ DNA polymerase, DEEPVENT™ DNApolymerase, LongAmp® Taq DNA polymerase, LongAmp® Hot Start Taq DNApolymerase, Crimson LongAmp® Taq DNA polymerase, Crimson Taq DNApolymerase, OneTaq® DNA polymerase, OneTaq® Quick-Load® DNA polymerase,Hemo KlenTaq® DNA polymerase, REDTaq® DNA polymerase, Phusion® DNApolymerase, Phusion® High-Fidelity DNA polymerase, Platinum Pfx DNApolymerase, AccuPrime Pfx DNA polymerase, Phi29 DNA polymerase, Klenowfragment, Pwo DNA polymerase, Pfu DNA polymerase, T4 DNA polymerase andT7 DNA polymerase enzymes.

The term “DNA polymerase” includes not only naturally-occurring enzymesbut also all modified derivatives thereof, including also derivatives ofnaturally-occurring DNA polymerase enzymes. For instance, in someembodiments, the DNA polymerase can have been modified to remove 5′-3′exonuclease activity. Sequence-modified derivatives or mutants of DNApolymerase enzymes that can be used include, but are not limited to,mutants that retain at least some of the functional, e.g. DNA polymeraseactivity of the wild-type sequence. Mutations can affect the activityprofile of the enzymes, e.g. enhance or reduce the rate ofpolymerization, under different reaction conditions, e.g. temperature,template concentration, primer concentration, etc. Mutations orsequence-modifications can also affect the exonuclease activity and/orthermostability of the enzyme.

In some embodiments, PCR amplification can include reactions such as,but not limited to, a strand-displacement amplification reaction, arolling circle amplification reaction, a ligase chain reaction, atranscription-mediated amplification reaction, an isothermalamplification reaction, and/or a loop-mediated amplification reaction.

In some embodiments, PCR amplification uses a single primer that iscomplementary to the 3′ tag of target DNA fragments. In someembodiments, PCR amplification uses a first and a second primer, whereat least a 3′ end portion of the first primer is complementary to atleast a portion of the 3′ tag of the target nucleic acid fragments, andwhere at least a 3′ end portion of the second primer exhibits thesequence of at least a portion of the 5′ tag of the target nucleic acidfragments. In some embodiments, a 5′ end portion of the first primer isnon-complementary to the 3′ tag of the target nucleic acid fragments,and a 5′ end portion of the second primer does not exhibit the sequenceof at least a portion of the 5′ tag of the target nucleic acidfragments. In some embodiments, the first primer includes a firstuniversal sequence and/or the second primer includes a second universalsequence.

In some embodiments (e.g., when the PCR amplification amplifies capturedDNA), the PCR amplification products can be ligated to additionalsequences using a DNA ligase enzyme. The DNA ligase activity can beprovided by one or more distinct DNA ligase enzymes. In someembodiments, the DNA ligase enzyme is from a bacterium, e.g., the DNAligase enzyme is a bacterial DNA ligase enzyme. In some embodiments, theDNA ligase enzyme is from a virus (e.g., a bacteriophage). For instance,the DNA ligase can be T4 DNA ligase. Other enzymes appropriate for theligation step include, but are not limited to, Tth DNA ligase, Taq DNAligase, Thermococcus sp. (strain 9oN) DNA ligase (9oNTM DNA ligase,available from New England Biolabs, Ipswich, MA), and Ampligase™(available from Epicentre Biotechnologies, Madison, WI). Derivatives,e.g. sequence-modified derivatives, and/or mutants thereof, can also beused.

In some embodiments, genetic material is amplified by reversetranscription polymerase chain reaction (RT-PCR). The desired reversetranscriptase activity can be provided by one or more distinct reversetranscriptase enzymes, suitable examples of which include, but are notlimited to: M-MLV, MuLV, AMV, HIV, ArrayScript™, MultiScribe™,ThermoScript™, and SuperScript® I, II, III, and IV enzymes. “Reversetranscriptase” includes not only naturally occurring enzymes, but allsuch modified derivatives thereof, including also derivatives ofnaturally-occurring reverse transcriptase enzymes.

In addition, reverse transcription can be performed usingsequence-modified derivatives or mutants of M-MLV, MuLV, AMV, and HIVreverse transcriptase enzymes, including mutants that retain at leastsome of the functional, e.g. reverse transcriptase, activity of thewild-type sequence. The reverse transcriptase enzyme can be provided aspart of a composition that includes other components, e.g. stabilizingcomponents that enhance or improve the activity of the reversetranscriptase enzyme, such as RNase inhibitor(s), inhibitors ofDNA-dependent DNA synthesis, e.g. actinomycin D. Many sequence-modifiedderivative or mutants of reverse transcriptase enzymes, e.g. M-MLV, andcompositions including unmodified and modified enzymes are commerciallyavailable, e.g. ArrayScript™, MultiScribe™, ThermoScript™, andSuperScript® I, II, III, and IV enzymes.

Certain reverse transcriptase enzymes (e.g. Avian Myeloblastosis Virus(AMV) Reverse Transcriptase and Moloney Murine Leukemia Virus (M-MuLV,MMLV) Reverse Transcriptase) can synthesize a complementary DNA strandusing both RNA (cDNA synthesis) and single-stranded DNA (ssDNA) as atemplate. Thus, in some embodiments, the reverse transcription reactioncan use an enzyme (reverse transcriptase) that is capable of using bothRNA and ssDNA as the template for an extension reaction, e.g. an AMV orMMLV reverse transcriptase.

In some embodiments, the quantification of RNA and/or DNA is carried outby real-time PCR (also known as quantitative PCR or qPCR), usingtechniques well known in the art, such as but not limited to “TAQMAN™”or “SYBR®”, or on capillaries (“LightCycler® Capillaries”). In someembodiments, the quantification of genetic material is determined byoptical absorbance and with real-time PCR. In some embodiments, thequantification of genetic material is determined by digital PCR. In someembodiments, the genes analyzed can be compared to a reference nucleicacid extract (DNA and RNA) corresponding to the expression (mRNA) andquantity (DNA) in order to compare expression levels of the targetnucleic acids.

(XIV) Antibody

An “antibody” is a polypeptide molecule that recognizes and binds to acomplementary target antigen. Antibodies typically have a molecularstructure shape that resembles a Y shape. Naturally-occurringantibodies, referred to as immunoglobulins, belong to one of theimmunoglobulin classes IgG, IgM, IgA, IgD, and IgE. Antibodies can alsobe produced synthetically. For example, recombinant antibodies, whichare monoclonal antibodies, can be synthesized using synthetic genes byrecovering the antibody genes from source cells, amplifying into anappropriate vector, and introducing the vector into a host to cause thehost to express the recombinant antibody. In general, recombinantantibodies can be cloned from any species of antibody-producing animalusing suitable oligonucleotide primers and/or hybridization probes.Recombinant techniques can be used to generate antibodies and antibodyfragments, including non-endogenous species.

Synthetic antibodies can be derived from non-immunoglobulin sources. Forexample, antibodies can be generated from nucleic acids (e.g.,aptamers), and from non-immunoglobulin protein scaffolds (such aspeptide aptamers) into which hypervariable loops are inserted to formantigen binding sites. Synthetic antibodies based on nucleic acids orpeptide structures can be smaller than immunoglobulin-derivedantibodies, leading to greater tissue penetration.

Antibodies can also include affimer proteins, which are affinityreagents that typically have a molecular weight of about 12-14 kDa.Affimer proteins generally bind to a target (e.g., a target protein)with both high affinity and specificity. Examples of such targetsinclude, but are not limited to, ubiquitin chains, immunoglobulins, andC-reactive protein. In some embodiments, affimer proteins are derivedfrom cysteine protease inhibitors, and include peptide loops and avariable N-terminal sequence that provides the binding site.

Antibodies can also include single domain antibodies (V_(H)H domains andVNAR domains), scFvs, and Fab fragments.

(XV) Affinity Group

An “affinity group” is a molecule or molecular moiety which has a highaffinity or preference for associating or binding with another specificor particular molecule or moiety. The association or binding withanother specific or particular molecule or moiety can be via anon-covalent interaction, such as hydrogen bonding, ionic forces, andvan der Waals interactions. An affinity group can, for example, bebiotin, which has a high affinity or preference to associate or bind tothe protein avidin or streptavidin. An affinity group, for example, canalso refer to avidin or streptavidin which has an affinity to biotin.Other examples of an affinity group and specific or particular moleculeor moiety to which it binds or associates with include, but are notlimited to, antibodies or antibody fragments and their respectiveantigens, such as digoxigenin and anti-digoxigenin antibodies, lectin,and carbohydrates (e.g., a sugar, a monosaccharide, a disaccharide, or apolysaccharide), and receptors and receptor ligands.

Any pair of affinity group and its specific or particular molecule ormoiety to which it binds or associates with can have their rolesreversed, for example, such that between a first molecule and a secondmolecule, in a first instance the first molecule is characterized as anaffinity group for the second molecule, and in a second instance thesecond molecule is characterized as an affinity group for the firstmolecule.

(XVI) Label, Detectable Label, and Optical Label

The terms “detectable label,” “optical label,” and “label” are usedinterchangeably herein to refer to a directly or indirectly detectablemoiety that is associated with (e.g., conjugated to) a molecule to bedetected, e.g., a capture probe or analyte. The detectable label can bedirectly detectable by itself (e.g., radioisotope labels or fluorescentlabels) or, in the case of an enzymatic label, can be indirectlydetectable, e.g., by catalyzing chemical alterations of a substratecompound or composition, which substrate compound or composition isdirectly detectable. Detectable labels can be suitable for small scaledetection and/or suitable for high-throughput screening. As such,suitable detectable labels include, but are not limited to,radioisotopes, fluorophores, chemiluminescent compounds, bioluminescentcompounds, and dyes.

The detectable label can be qualitatively detected (e.g., optically orspectrally), or it can be quantified. Qualitative detection generallyincludes a detection method in which the existence or presence of thedetectable label is confirmed, whereas quantifiable detection generallyincludes a detection method having a quantifiable (e.g., numericallyreportable) value such as an intensity, duration, polarization, and/orother properties. In some embodiments, the detectable label is bound toa feature or to a capture probe associated with a feature. For example,detectably labeled features can include a fluorescent, a colorimetric,or a chemiluminescent label attached to a bead (see, for example,Rajeswari et al., J. Microbiol Methods 139:22-28, 2017, and Forcucci etal., J. Biomed Opt. 10:105010, 2015, the entire contents of each ofwhich are incorporated herein by reference).

In some embodiments, a plurality of detectable labels can be attached toa feature, capture probe, or composition to be detected. For example,detectable labels can be incorporated during nucleic acid polymerizationor amplification (e.g., Cy5®-labelled nucleotides, such as Cy5®-dCTP).Any suitable detectable label can be used. In some embodiments, thedetectable label is a fluorophore. For example, the fluorophore can befrom a group that includes: 7-AAD (7-Aminoactinomycin D), AcridineOrange (+DNA), Acridine Orange (+RNA), Alexa Fluor® 350, Alexa Fluor®430, Alexa Fluor® 488, Alexa Fluor® 532, Alexa Fluor® 546, Alexa Fluor®555, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 633, Alexa Fluor®647, Alexa Fluor® 660, Alexa Fluor® 680, Alexa Fluor® 700, Alexa Fluor®750, Allophycocyanin (APC), AMCA / AMCA-X, 7-Aminoactinomycin D (7-AAD),7- Amino-4-methylcoumarin, 6-Aminoquinoline, Aniline Blue, ANS, APC-Cy7,ATTO-TAG™ CBQCA, ATTO-TAG™ FQ, Auramine O-Feulgen, BCECF (high pH), BFP(Blue Fluorescent Protein), BFP / GFP FRET, BOBO™-1 / BO-PRO™-1, BOBO™-3/ BO-PRO™-3, BODIPY® FL, BODIPY® TMR, BODIPY® TR-X, BODIPY® 530/550,BODIPY® 558/568, BODIPY® 564/570, BODIPY® 581/591, BODIPY® 630/650-X,BODIPY® 650-665-X, BTC, Calcein, Calcein Blue, Calcium Crimson™, CalciumGreen-1™, Calcium Orange™, Calcofluor® White, 5-Carboxyfluoroscein(5-FAM), 5-Carboxynaphthofluoroscein, 6-Carboxyrhodamine 6G,5-Carboxytetramethylrhodamine (5-TAMRA), Carboxy-X-rhodamine (5-ROX),Cascade Blue®, Cascade Yellow™, CCF2 (GeneBLAzer™), CFP (CyanFluorescent Protein), CFP / YFP FRET, Chromomycin A3, Cl-NERF (low pH),CPM, 6-CR 6G, CTC Formazan, Cy2®, Cy3®, Cy3.5®, Cy5®, Cy5.5®, Cy7®,Cychrome (PE-Cy5), Dansylamine, Dansyl cadaverine, Dansylchloride, DAPI,Dapoxyl, DCFH, DHR, DiA (4-Di-16-ASP), DiD (DilC18(5)), DIDS, Dil(DilC18(3)), DiO (DiOC18(3)), DiR (DilC18(7)), Di-4 ANEPPS, Di-8 ANEPPS,DM-NERF (4.5-6.5 pH), DsRed (Red Fluorescent Protein), EBFP, ECFP, EGFP,ELF® -97 alcohol, Eosin, Erythrosin, Ethidium bromide, Ethidiumhomodimer-1 (EthD-1), Europium (III) Chloride, 5-FAM(5-Carboxyfluorescein), Fast Blue, Fluorescein-dT phosphoramidite, FITC,Fluo-3, Fluo-4, FluorX®, Fluoro-Gold™ (high pH), Fluoro-Gold™ (low pH),Fluoro-Jade, FM® 1-43, Fura-2 (high calcium), Fura-2 / BCECF, Fura Red™(high calcium), Fura Red™ / Fluo-3, GeneBLAzer™ (CCF2), GFP Red Shifted(rsGFP), GFP Wild Type, GFP / BFP FRET, GFP / DsRed FRET, Hoechst 33342& 33258, 7-Hydroxy-4-methylcoumarin (pH 9), 1,5 IAEDANS, Indo-1 (highcalcium), Indo-1 (low calcium), Indodicarbocyanine, Indotricarbocyanine,JC-1, 6-JOE, JOJO™-1 / JO-PRO™-1, LDS 751 (+DNA), LDS 751 (+RNA),LOLO™-1 / LO-PRO™-1, Lucifer Yellow, LysoSensor™ Blue (pH 5),LysoSensor™ Green (pH 5), LysoSensor™ Yellow/Blue (pH 4.2), LysoTracker®Green, LysoTracker® Red, LysoTracker® Yellow, Mag-Fura-2, Mag-Indo-1,Magnesium Green™, Marina Blue®, 4-Methylumbelliferone, Mithramycin,MitoTracker® Green, MitoTracker® Orange, MitoTracker® Red, NBD (amine),Nile Red, Oregon Green® 488, Oregon Green® 500, Oregon Green® 514,Pacific Blue, PBF1, PE (R-phycoerythrin), PE-Cy5, PE-Cy7, PE-Texas Red,PerCP (Peridinin chlorphyll protein), PerCP-Cy5.5 (TruRed), PharRed(APC-Cy7), C-phycocyanin, R-phycocyanin, R-phycoerythrin (PE), PI(Propidium Iodide), PKH26, PKH67, POPO™-1 / PO-PRO™-1, POPO™-3 /PO-PRO™-3, Propidium Iodide (PI), PyMPO, Pyrene, Pyronin Y, Quantam Red(PE-Cy5), Quinacrine Mustard, R670 (PE-Cy5), Red 613 (PE-Texas Red), RedFluorescent Protein (DsRed), Resorufin, RH 414, Rhod-2, Rhodamine B,Rhodamine Green™, Rhodamine Red™, Rhodamine Phalloidin, Rhodamine 110,Rhodamine 123, 5-ROX (carboxy-X-rhodamine), S65A, S65C, S65L, S65T,SBFI, SITS, SNAFL®-1 (high pH), SNAFL®-2, SNARF®-1 (high pH), SNARF®-1(low pH), Sodium Green™, SpectrumAqua®, SpectrumGreen® #1,SpectrumGreen® #2, SpectrumOrange®, SpectrumRed®, SYTO® 11, SYTO® 13,SYTO® 17, SYTO® 45, SYTOX® Blue, SYTOX® Green, SYTOX® Orange, 5-TAMRA(5-Carboxytetramethylrhodamine), Tetramethylrhodamine (TRITC), TexasRed® / Texas Red®-X, Texas Red®-X (NHS Ester), Thiadicarbocyanine,Thiazole Orange, TOTO®-1 / TO-PRO®-1, TOTO®-3 / TO-PRO®-3, TO-PRO®-5,Tri-color (PE-Cy5), TRITC (Tetramethylrhodamine), TruRed (PerCP-Cy5.5),WW 781, X-Rhodamine (XRITC), Y66F, Y66H, Y66W, YFP (Yellow FluorescentProtein), YOYO®-1 / YO-PRO®-1, YOYO®-3 / YO-PRO®-3, 6-FAM (Fluorescein),6-FAM (NHS Ester), 6-FAM (Azide), HEX, TAMRA (NHS Ester), Yakima Yellow,MAX, TET, TEX615, ATTO 488, ATTO 532, ATTO 550, ATTO 565, ATTO Rho101,ATTO 590, ATTO 633, ATTO 647N, TYE 563, TYE 665, TYE 705, 5′ IRDye® 700,5′ IRDye® 800, 5′ IRDye® 800CW (NHS Ester), WellRED D4 Dye, WellRED D3Dye, WellRED D2 Dye, Lightcycler® 640 (NHS Ester), and Dy 750 (NHSEster).

As mentioned above, in some embodiments, a detectable label is orincludes a luminescent or chemiluminescent moiety. Commonluminescent/chemiluminescent moieties include, but are not limited to,peroxidases such as horseradish peroxidase (HRP), soybean peroxidase(SP), alkaline phosphatase, and luciferase. These protein moieties cancatalyze chemiluminescent reactions given the appropriate substrates(e.g., an oxidizing reagent plus a chemiluminescent compound. A numberof compound families are known to provide chemiluminescence under avariety of conditions. Non-limiting examples of chemiluminescentcompound families include 2,3-dihydro-1,4-phthalazinedione luminol,5-amino-6,7,8-trimethoxy-and the dimethylamino[ca]benz analog. Thesecompounds can luminesce in the presence of alkaline hydrogen peroxide orcalcium hypochlorite and base. Other examples of chemiluminescentcompound families include, e.g., 2,4,5-triphenylimidazoles,para-dimethylamino and - methoxy substituents, oxalates such as oxalylactive esters, p-nitrophenyl, N-alkyl acridinum esters, luciferins,lucigenins, or acridinium esters.

(XVII) Template Switching Oligonucleotide

A “template switching oligonucleotide” is an oligonucleotide thathybridizes to untemplated nucleotides added by a reverse transcriptase(e.g., enzyme with terminal transferase activity) during reversetranscription. In some embodiments, a template switching oligonucleotidehybridizes to untemplated poly(C) nucleotides added by a reversetranscriptase. In some embodiments, the template switchingoligonucleotide adds a common 5′ sequence to full-length cDNA that isused for cDNA amplification.

In some embodiments, the template switching oligonucleotide adds acommon sequence onto the 5′ end of the RNA being reverse transcribed.For example, a template switching oligonucleotide can hybridize tountemplated poly(C) nucleotides added onto the end of a cDNA moleculeand provide a template for the reverse transcriptase to continuereplication to the 5′ end of the template switching oligonucleotide,thereby generating full-length cDNA ready for further amplification. Insome embodiments, once a full-length cDNA molecule is generated, thetemplate switching oligonucleotide can serve as a primer in a cDNAamplification reaction.

In some embodiments, a template switching oligonucleotide is addedbefore, contemporaneously with, or after a reverse transcription, orother terminal transferase-based reaction. In some embodiments, atemplate switching oligonucleotide is included in the capture probe. Incertain embodiments, methods of sample analysis using template switchingoligonucleotides can involve the generation of nucleic acid productsfrom analytes of the tissue sample, followed by further processing ofthe nucleic acid products with the template switching oligonucleotide.

Template switching oligonucleotides can include a hybridization regionand a template region. The hybridization region can include any sequencecapable of hybridizing to the target. In some embodiments, thehybridization region can, e.g., include a series of G bases tocomplement the overhanging C bases at the 3′ end of a cDNA molecule. Theseries of G bases can include 1 G base, 2 G bases, 3 G bases, 4 G bases,5 G bases, or more than 5 G bases. The template sequence can include anysequence to be incorporated into the cDNA. In other embodiments, thehybridization region can include at least one base in addition to atleast one G base. In other embodiments, the hybridization can includebases that are not a G base. In some embodiments, the template regionincludes at least 1 (e.g., at least 2, 3, 4, 5 or more) tag sequencesand/or functional sequences. In some embodiments, the template regionand hybridization region are separated by a spacer.

In some embodiments, the template regions include a barcode sequence.The barcode sequence can act as a spatial barcode and/or as a uniquemolecular identifier. Template switching oligonucleotides can includedeoxyribonucleic acids; ribonucleic acids; modified nucleic acidsincluding 2-aminopurine, 2,6-diaminopurine (2-amino-dA), inverted dT,5-methyl dC, 2′-deoxyInosine, Super T (5-hydroxybutynl-2′-deoxyuridine),Super G (8-aza-7-deazaguanosine), locked nucleic acids (LNAs), unlockednucleic acids (UNAs, e.g., UNA-A, UNA-U, UNA-C, UNA-G), Iso-dG, Iso-dC,2′ fluoro bases (e.g., Fluoro C, Fluoro U, Fluoro A, and Fluoro G), orany combination of the foregoing.

In some embodiments, the length of a template switching oligonucleotidecan be at least about 1, 2, 10, 20, 50, 75, 100, 150, 200, or 250nucleotides or longer. In some embodiments, the length of a templateswitching oligonucleotide can be at most about 2, 10, 20, 50, 100, 150,200, or 250 nucleotides or longer.

(XVIII) Splint Oligonucleotide

A “splint oligonucleotide” is an oligonucleotide that, when hybridizedto other polynucleotides, acts as a “splint” to position thepolynucleotides next to one another so that they can be ligatedtogether. In some embodiments, the splint oligonucleotide is DNA or RNA.The splint oligonucleotide can include a nucleotide sequence that ispartially complimentary to nucleotide sequences from two or moredifferent oligonucleotides. In some embodiments, the splintoligonucleotide assists in ligating a “donor” oligonucleotide and an“acceptor” oligonucleotide. In general, an RNA ligase, a DNA ligase, oranother other variety of ligase is used to ligate two nucleotidesequences together

In some embodiments, the splint oligonucleotide is between 10 and 50oligonucleotides in length, e.g., between 10 and 45, 10 and 40, 10 and35, 10 and 30, 10 and 25, or 10 and 20 oligonucleotides in length. Insome embodiments, the splint oligonucleotide is between 15 and 50, 15and 45, 15 and 40, 15 and 35, 15 and 30, 15 and 30, or 15 and 25nucleotides in length.

(C) Analytes

The apparatus, systems, methods, and compositions described in thisdisclosure can be used to detect and analyze a wide variety of differentanalytes. For the purpose of this disclosure, an “analyte” can includeany biological substance, structure, moiety, or component to beanalyzed. The term “target” can similarly refer to an analyte ofinterest.

Analytes can be broadly classified into one of two groups: nucleic acidanalytes, and non-nucleic acid analytes. Examples of non-nucleic acidanalytes include, but are not limited to, lipids, carbohydrates,peptides, proteins, glycoproteins (N-linked or O-linked), lipoproteins,phosphoproteins, specific phosphorylated or acetylated variants ofproteins, amidation variants of proteins, hydroxylation variants ofproteins, methylation variants of proteins, ubiquitylation variants ofproteins, sulfation variants of proteins, viral coat proteins,extracellular and intracellular proteins, antibodies, and antigenbinding fragments. In some embodiments, the analyte can be an organelle(e.g., nuclei or mitochondria).

Cell surface features corresponding to analytes can include, but are notlimited to, a receptor, an antigen, a surface protein, a transmembraneprotein, a cluster of differentiation protein, a protein channel, aprotein pump, a carrier protein, a phospholipid, a glycoprotein, aglycolipid, a cell-cell interaction protein complex, anantigen-presenting complex, a major histocompatibility complex, anengineered T-cell receptor, a T-cell receptor, a B-cell receptor, achimeric antigen receptor, an extracellular matrix protein, aposttranslational modification (e.g., phosphorylation, glycosylation,ubiquitination, nitrosylation, methylation, acetylation or lipidation)state of a cell surface protein, a gap junction, and an adherensjunction.

Analytes can be derived from a specific type of cell and/or a specificsub-cellular region. For example, analytes can be derived from cytosol,from cell nuclei, from mitochondria, from microsomes, and moregenerally, from any other compartment, organelle, or portion of a cell.Permeabilizing agents that specifically target certain cell compartmentsand organelles can be used to selectively release analytes from cellsfor analysis.

Examples of nucleic acid analytes include DNA analytes such as genomicDNA, methylated DNA, specific methylated DNA sequences, fragmented DNA,mitochondrial DNA, in situ synthesized PCR products, and RNA/DNAhybrids.

Examples of nucleic acid analytes also include RNA analytes such asvarious types of coding and non-coding RNA. Examples of the differenttypes of RNA analytes include messenger RNA (mRNA), ribosomal RNA(rRNA), transfer RNA (tRNA), microRNA (miRNA), and viral RNA. The RNAcan be a transcript (e.g., present in a tissue section). The RNA can besmall (e.g., less than 200 nucleic acid bases in length) or large (e.g.,RNA greater than 200 nucleic acid bases in length). Small RNAs mainlyinclude 5.8S ribosomal RNA (rRNA), 5S rRNA, transfer RNA (tRNA),microRNA (miRNA), small interfering RNA (siRNA), small nucleolar RNA(snoRNAs), Piwi-interacting RNA (piRNA), tRNA-derived small RNA (tsRNA),and small rDNA-derived RNA (srRNA). The RNA can be double-stranded RNAor single-stranded RNA. The RNA can be circular RNA. The RNA can be abacterial rRNA (e.g., 16s rRNA or 23s rRNA).

Additional examples of analytes include mRNA and cell surface features(e.g., using the labelling agents described herein), mRNA andintracellular proteins (e.g., transcription factors), mRNA and cellmethylation status, mRNA and accessible chromatin (e.g., ATAC-seq,DNase-seq, and/or MNase-seq), mRNA and metabolites (e.g., using thelabelling agents described herein), a barcoded labelling agent (e.g.,the oligonucleotide tagged antibodies described herein) and a V(D)Jsequence of an immune cell receptor (e.g., T-cell receptor), mRNA and aperturbation agent (e.g., a CRISPR crRNA/sgRNA, TALEN, zinc fingernuclease, and/or antisense oligonucleotide as described herein).

Analytes can include a nucleic acid molecule with a nucleic acidsequence encoding at least a portion of a V(D)J sequence of an immunecell receptor (e.g., a TCR or BCR). In some embodiments, the nucleicacid molecule is cDNA first generated from reverse transcription of thecorresponding mRNA, using a poly(T) containing primer. The generatedcDNA can then be barcoded using a capture probe, featuring a barcodesequence (and optionally, a UMI sequence) that hybridizes with at leasta portion of the generated cDNA. In some embodiments, a templateswitching oligonucleotide hybridizes to a poly(C) tail added to a 3′ endof the cDNA by a reverse transcriptase enzyme. The original mRNAtemplate and template switching oligonucleotide can then be denaturedfrom the cDNA and the barcoded capture probe can then hybridize with thecDNA and a complement of the cDNA generated. Additional methods andcompositions suitable for barcoding cDNA generated from mRNA transcriptsincluding those encoding V(D)J regions of an immune cell receptor and/orbarcoding methods and composition including a template switcholigonucleotide are described in PCT Patent ApplicationPCT/US2017/057269, filed Oct. 18, 2017, and U.S. Pat. Application SerialNo. 15/825,740, filed Nov. 29, 2017, both of which are incorporatedherein by reference in their entireties. V(D)J analysis can also becompleted with the use of one or more labelling agents that bind toparticular surface features of immune cells and associated with barcodesequences. The one or more labelling agents can include an MHC or MHCmultimer.

As described above, the analyte can include a nucleic acid capable offunctioning as a component of a gene editing reaction, such as, forexample, clustered regularly interspaced short palindromic repeats(CRISPR)-based gene editing. Accordingly, the capture probe can includea nucleic acid sequence that is complementary to the analyte (e.g., asequence that can hybridize to the CRISPR RNA (crRNA), single guide RNA(sgRNA), or an adapter sequence engineered into a crRNA or sgRNA).

In certain embodiments, an analyte can be extracted from a live cell.Processing conditions can be adjusted to ensure that a biological sampleremains live during analysis, and analytes are extracted from (orreleased from) live cells of the sample. Live cell-derived analytes canbe obtained only once from the sample, or can be obtained at intervalsfrom a sample that continues to remain in viable condition.

In general, the systems, apparatus, methods, and compositions can beused to analyze any number of analytes. For example, the number ofanalytes that are analyzed can be at least about 2, at least about 3, atleast about 4, at least about 5, at least about 6, at least about 7, atleast about 8, at least about 9, at least about 10, at least about 11,at least about 12, at least about 13, at least about 14, at least about15, at least about 20, at least about 25, at least about 30, at leastabout 40, at least about 50, at least about 100, at least about 1,000,at least about 10,000, at least about 100,000 or more different analytespresent in a region of the sample or within an individual feature of thesubstrate. Methods for performing multiplexed assays to analyze two ormore different analytes will be discussed in a subsequent section ofthis disclosure.

(D) Biological Samples (I) Types of Biological Samples

A “biological sample” is obtained from the subject for analysis usingany of a variety of techniques including, but not limited to, biopsy,surgery, and laser capture microscopy (LCM), and generally includescells and/or other biological material from the subject. In addition tothe subjects described above, a biological sample can also be obtainedfrom a prokaryote such as a bacterium, e.g., Escherichia coli,Staphylococci or Mycoplasma pneumoniae; an archaea; a virus such asHepatitis C virus or human immunodeficiency virus; or a viroid. Abiological sample can be obtained from non-mammalian organisms (e.g., aplants, an insect, an arachnid, a nematode, a fungi, or an amphibian). Abiological sample can also be obtained from a eukaryote, such as apatient derived organoid (PDO) or patient derived xenograft (PDX).Subjects from which biological samples can be obtained can be healthy orasymptomatic individuals, individuals that have or are suspected ofhaving a disease (e.g., a patient with a disease such as cancer) or apredisposition to a disease, and/or individuals that are in need oftherapy or suspected of needing therapy.

The biological sample can include any number of macromolecules, forexample, cellular macromolecules and organelles (e.g., mitochondria andnuclei). The biological sample can be a nucleic acid sample and/orprotein sample. The biological sample can be a carbohydrate sample or alipid sample. The biological sample can be obtained as a tissue sample,such as a tissue section, biopsy, a core biopsy, needle aspirate, orfine needle aspirate. The sample can be a fluid sample, such as a bloodsample, urine sample, or saliva sample. The sample can be a skin sample,a colon sample, a cheek swab, a histology sample, a histopathologysample, a plasma or serum sample, a tumor sample, living cells, culturedcells, a clinical sample such as, for example, whole blood orblood-derived products, blood cells, or cultured tissues or cells,including cell suspensions.

Cell-free biological samples can include extracellular polynucleotides.Extracellular polynucleotides can be isolated from a bodily sample,e.g., blood, plasma, serum, urine, saliva, mucosal excretions, sputum,stool, and tears.

Biological samples can be derived from a homogeneous culture orpopulation of the subjects or organisms mentioned herein oralternatively from a collection of several different organisms, forexample, in a community or ecosystem.

Biological samples can include one or more diseased cells. A diseasedcell can have altered metabolic properties, gene expression, proteinexpression, and/or morphologic features. Examples of diseases includeinflammatory disorders, metabolic disorders, nervous system disorders,and cancer. Cancer cells can be derived from solid tumors, hematologicalmalignancies, cell lines, or obtained as circulating tumor cells.

Biological samples can also include fetal cells. For example, aprocedure such as amniocentesis can be performed to obtain a fetal cellsample from maternal circulation. Sequencing of fetal cells can be usedto identify any of a number of genetic disorders, including, e.g.,aneuploidy such as Down’s syndrome, Edwards syndrome, and Patausyndrome. Further, cell surface features of fetal cells can be used toidentify any of a number of disorders or diseases.

Biological samples can also include immune cells. Sequence analysis ofthe immune repertoire of such cells, including genomic, proteomic, andcell surface features, can provide a wealth of information to facilitatean understanding the status and function of the immune system. By way ofexample, determining the status (e.g., negative or positive) of minimalresidue disease (MRD) in a multiple myeloma (MM) patient followingautologous stem cell transplantation is considered a predictor of MRD inthe MM patient (see, e.g., U.S. Pat. Application Publication No.2018/0156784, the entire contents of which are incorporated herein byreference).

Examples of immune cells in a biological sample include, but are notlimited to, B cells, T cells (e.g., cytotoxic T cells, natural killer Tcells, regulatory T cells, and T helper cells), natural killer cells,cytokine induced killer (CIK) cells, myeloid cells, such as granulocytes(basophil granulocytes, eosinophil granulocytes, neutrophilgranulocytes/hypersegmented neutrophils), monocytes/macrophages, mastcells, thrombocytes/megakaryocytes, and dendritic cells.

As discussed above, a biological sample can include a single analyte ofinterest, or more than one analyte of interest. Methods for performingmultiplexed assays to analyze two or more different analytes in a singlebiological sample will be discussed in a subsequent section of thisdisclosure.

(II) Preparation of Biological Samples

A variety of steps can be performed to prepare a biological sample foranalysis. Except where indicated otherwise, the preparative stepsdescribed below can generally be combined in any manner to appropriatelyprepare a particular sample for analysis.

Tissue Sectioning

A biological sample can be harvested from a subject (e.g., via surgicalbiopsy, whole subject sectioning) or grown in vitro on a growthsubstrate or culture dish as a population of cells, and prepared foranalysis as a tissue slice or tissue section. Grown samples may besufficiently thin for analysis without further processing steps.Alternatively, grown samples, and samples obtained via biopsy orsectioning, can be prepared as thin tissue sections using a mechanicalcutting apparatus such as a vibrating blade microtome. As anotheralternative, in some embodiments, a thin tissue section can be preparedby applying a touch imprint of a biological sample to a suitablesubstrate material.

The thickness of the tissue section can be a fraction of (e.g., lessthan 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1) the maximumcross-sectional dimension of a cell. However, tissue sections having athickness that is larger than the maximum cross-section cell dimensioncan also be used. For example, cryostat sections can be used, which canbe, e.g., 10-20 micrometers thick.

More generally, the thickness of a tissue section typically depends onthe method used to prepare the section and the physical characteristicsof the tissue, and therefore sections having a wide variety of differentthicknesses can be prepared and used. For example, the thickness of thetissue section can be at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.7, 1.0, 1.5,2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 20, 30, 40, or 50micrometers. Thicker sections can also be used if desired or convenient,e.g., at least 70, 80, 90, or 100 micrometers or more. Typically, thethickness of a tissue section is between 1-100 micrometers, 1-50micrometers, 1-30 micrometers, 1-25 micrometers, 1-20 micrometers, 1-15micrometers, 1-10 micrometers, 2-8 micrometers, 3-7 micrometers, or 4-6micrometers, but as mentioned above, sections with thicknesses larger orsmaller than these ranges can also be analysed.

Multiple sections can also be obtained from a single biological sample.For example, multiple tissue sections can be obtained from a surgicalbiopsy sample by performing serial sectioning of the biopsy sample usinga sectioning blade. Spatial information among the serial sections can bepreserved in this manner, and the sections can be analysed successivelyto obtain three-dimensional information about the biological sample.

Freezing

In some embodiments, the biological sample (e.g., a tissue section asdescribed above) can be prepared by deep freezing at a temperaturesuitable to maintain or preserve the integrity (e.g., the physicalcharacteristics) of the tissue structure. Such a temperature can be,e.g., less than -20° C., or less than -25° C., -30° C., -40° C., -50°C., -60° C., -70° C., -80° C. -90° C., -100° C., -110° C., -120° C.,-130° C., -140° C., -150° C., -160° C., -170° C., -180° C., -190° C., or-200° C. The frozen tissue sample can be sectioned, e.g., thinly sliced,onto a substrate surface using any number of suitable methods. Forexample, a tissue sample can be prepared using a chilled microtome(e.g., a cryostat) set at a temperature suitable to maintain both thestructural integrity of the tissue sample and the chemical properties ofthe nucleic acids in the sample. Such a temperature can be, e.g., lessthan -15° C., less than -20° C., or less than -25° C.

Formalin Fixation and Paraffin Embedding

In some embodiments, the biological sample can be prepared usingformalin-fixation and paraffin-embedding (FFPE), which are establishedmethods. In some embodiments, cell suspensions and other non-tissuesamples can be prepared using formalin-fixation and paraffin-embedding.Following fixation of the sample and embedding in a paraffin or resinblock, the sample can be sectioned as described above. Prior toanalysis, the paraffin-embedding material can be removed from the tissuesection (e.g., deparaffinization) by incubating the tissue section in anappropriate solvent (e.g., xylene) followed by a rinse (e.g., 99.5%ethanol for 2 minutes, 96% ethanol for 2 minutes, and 70% ethanol for 2minutes).

Fixation

As an alternative to formalin fixation described above, a biologicalsample can be fixed in any of a variety of other fixatives to preservethe biological structure of the sample prior to analysis. For example, asample can be fixed via immersion in ethanol, methanol, acetone,paraformaldehyde-Triton, and combinations thereof.

In some embodiments, acetone fixation is used with fresh frozen samples,which can include, but are not limited to, cortex tissue, mouseolfactory bulb, human brain tumor, human post-mortem brain, and breastcancer samples. When acetone fixation is performed, pre-permeabilizationsteps (described below) may not be performed. Alternatively, acetonefixation can be performed in conjunction with permeabilization steps.

Embedding

As an alternative to paraffin embedding described above, a biologicalsample can be embedded in any of a variety of other embedding materialsto provide structural substrate to the sample prior to sectioning andother handling steps. In general, the embedding material is removedprior to analysis of tissue sections obtained from the sample. Suitableembedding materials include, but are not limited to, waxes, resins(e.g., methacrylate resins), epoxies, and agar.

Staining

To facilitate visualization, biological samples can be stained using awide variety of stains and staining techniques. In some embodiments, forexample, a sample can be stained using any number of stains, includingbut not limited to, acridine orange, Bismarck brown, carmine, coomassieblue, cresyl violet, DAPI, eosin, ethidium bromide, acid fuchsine,haematoxylin, Hoechst stains, iodine, methyl green, methylene blue,neutral red, Nile blue, Nile red, osmium tetroxide, propidium iodide,rhodamine, or safranine.

The sample can be stained using hematoxylin and eosin (H&E) stainingtechniques, using Papanicolaou staining techniques, Masson’s trichromestaining techniques, silver staining techniques, Sudan stainingtechniques, and/or using Periodic Acid Schiff (PAS) staining techniques.PAS staining is typically performed after formalin or acetone fixation.In some embodiments, the sample can be stained using Romanowsky stain,including Wright’s stain, Jenner’s stain, Can-Grunwald stain, Leishmanstain, and Giemsa stain.

In some embodiments, biological samples can be destained. Methods ofdestaining or discoloring a biological sample are known in the art, andgenerally depend on the nature of the stain(s) applied to the sample.For example, in some embodiments, one or more immunofluorescent stainsare applied to the sample via antibody coupling. Such stains can beremoved using techniques such as cleavage of disulfide linkages viatreatment with a reducing agent and detergent washing, chaotropic salttreatment, treatment with antigen retrieval solution, and treatment withan acidic glycine buffer. Methods for multiplexed staining anddestaining are described, for example, in Bolognesi et al., J.Histochem. Cytochem. 2017; 65(8): 431-444, Lin et al., Nat Commun. 2015;6:8390, Pirici et al., J. Histochem. Cytochem. 2009; 57:567-75, andGlass et al., J. Histochem. Cytochem. 2009; 57:899-905, the entirecontents of each of which are incorporated herein by reference.

Hydrogel Embedding

In some embodiments, the biological sample can be embedded in a hydrogelmatrix. Embedding the sample in this manner typically involvescontacting the biological sample with a hydrogel such that thebiological sample becomes surrounded by the hydrogel. For example, thesample can be embedded by contacting the sample with a suitable polymermaterial, and activating the polymer material to form a hydrogel. Insome embodiments, the hydrogel is formed such that the hydrogel isinternalized within the biological sample.

In some embodiments, the biological sample is immobilized in thehydrogel via cross-linking of the polymer material that forms thehydrogel. Cross-linking can be performed chemically and/orphotochemically, or alternatively by any other hydrogel-formation methodknown in the art.

The composition and application of the hydrogel-matrix to a biologicalsample typically depends on the nature and preparation of the biologicalsample (e.g., sectioned, non-sectioned, type of fixation). As oneexample, where the biological sample is a tissue section, thehydrogel-matrix can include a monomer solution and an ammoniumpersulfate (APS) initiator/tetramethylethylenediamine (TEMED)accelerator solution. As another example, where the biological sampleconsists of cells (e.g., cultured cells or cells disassociated from atissue sample), the cells can be incubated with the monomer solution andAPS/TEMED solutions. For cells, hydrogel-matrix gels are formed incompartments, including but not limited to devices used to culture,maintain, or transport the cells. For example, hydrogel-matrices can beformed with monomer solution plus APS/TEMED added to the compartment toa depth ranging from about 0.1 µm to about 2 mm.

Additional methods and aspects of hydrogel embedding of biologicalsamples are described for example in Chen et al., Science347(6221):543-548, 2015, the entire contents of which are incorporatedherein by reference.

Isometric Expansion

In some embodiments, a biological sample embedded in a hydrogel can beisometrically expanded. Isometric expansion methods that can be usedinclude hydration, a preparative step in expansion microscopy, asdescribed in Chen et al., Science 347(6221):543-548, 2015.

Isometric expansion can be performed by anchoring one or more componentsof a biological sample to a gel, followed by gel formation, proteolysis,and swelling. Isometric expansion of the biological sample can occurprior to immobilization of the biological sample on a substrate, orafter the biological sample is immobilized to a substrate. In someembodiments, the isometrically expanded biological sample can be removedfrom the substrate prior to contacting the substrate with captureprobes, as will be discussed in greater detail in a subsequent section.

In general, the steps used to perform isometric expansion of thebiological sample can depend on the characteristics of the sample (e.g.,thickness of tissue section, fixation, cross-linking), and/or theanalyte of interest (e.g., different conditions to anchor RNA, DNA, andprotein to a gel).

In some embodiments, proteins in the biological sample are anchored to aswellable gel such as a polyelectrolyte gel. An antibody can be directedto the protein before, after, or in conjunction with being anchored tothe swellable gel. DNA and/or RNA in a biological sample can also beanchored to the swellable gel via a suitable linker. Examples of suchlinkers include, but are not limited to, 6-((Acryloyl)amino) hexanoicacid (Acryloyl-X SE) (available from ThermoFisher, Waltham, MA),Label-IT Amine (available from MirusBio, Madison, WI) and Label X(described for example in Chen et al., Nat. Methods 13:679-684, 2016,the entire contents of which are incorporated herein by reference).

Isometric expansion of the sample can increase the spatial resolution ofthe subsequent analysis of the sample. The increased resolution inspatial profiling can be determined by comparison of an isometricallyexpanded sample with a sample that has not been isometrically expanded.

In some embodiments, a biological sample is isometrically expanded to asize at least 2x, 2.1x, 2.2x, 2.3x, 2.4x, 2.5x, 2.6x, 2.7x, 2.8x, 2.9x,3x, 3.1x, 3.2x, 3.3x, 3.4x, 3.5x, 3.6x, 3.7x, 3.8x, 3.9x, 4x, 4.1x,4.2x, 4.3x, 4.4x, 4.5x, 4.6x, 4.7x, 4.8x, or 4.9x its non-expanded size.In some embodiments, the sample is isometrically expanded to at least 2xand less than 20x of its non-expanded size.

Substrate Attachment

In some embodiments, the biological sample can be attached to asubstrate. Examples of substrates suitable for this purpose aredescribed in detail below. Attachment of the biological sample can beirreversible or reversible, depending upon the nature of the sample andsubsequent steps in the analytical method.

In certain embodiments, the sample can be attached to the substratereversibly by applying a suitable polymer coating to the substrate, andcontacting the sample to the polymer coating. The sample can then bedetached from the substrate using an organic solvent that at leastpartially dissolves the polymer coating. Hydrogels are examples ofpolymers that are suitable for this purpose.

More generally, in some embodiments, the substrate can be coated orfunctionalized with one or more substances to facilitate attachment ofthe sample to the substrate. Suitable substances that can be used tocoat or functionalize the substrate include, but are not limited to,lectins, poly-lysine, antibodies, and polysaccharides.

Disaggregation of Cells

In some embodiments, the biological sample corresponds to cells (e.g.,derived from a cell culture or a tissue sample). In a cell sample with aplurality of cells, individual cells can be naturally unaggregated. Forexample, the cells can be derived from a suspension of cells and/ordisassociated or disaggregated cells from a tissue or tissue section.

Alternatively, the cells in the sample may be aggregated, and may bedisaggregated into individual cells using, for example, enzymatic ormechanical techniques. Examples of enzymes used in enzymaticdisaggregation include, but are not limited to, dispase, collagenase,trypsin, and combinations thereof. Mechanical disaggregation can beperformed, for example, using a tissue homogenizer.

Suspended and Adherent Cells

In some embodiments, the biological sample can be derived from a cellculture grown in vitro. Samples derived from a cell culture can includeone or more suspension cells which are anchorage-independent within thecell culture. Examples of such cells include, but are not limited to,cell lines derived from hematopoietic cells, and from the following celllines: Colo205, CCRF-CEM, HL-60, K562, MOLT-4, RPMI-8226, SR, HOP-92,NCI-H322M, and MALME-3M.

Samples derived from a cell culture can include one or more adherentcells which grow on the surface of the vessel that contains the culturemedium. Non-limiting examples of adherent cells include DU145 (prostatecancer) cells, H295R (adrenocortical cancer) cells, HeLa (cervicalcancer) cells, KBM-7 (chronic myelogenous leukemia) cells, LNCaP(prostate cancer) cells, MCF-7 (breast cancer) cells, MDA-MB-468 (breastcancer) cells, PC3 (prostate cancer) cells, SaOS-2 (bone cancer) cells,SH-SY5Y (neuroblastoma, cloned from a myeloma) cells, T-47D (breastcancer) cells, THP-1 (acute myeloid leukemia) cells, U87 (glioblastoma)cells, National Cancer Institute’s 60 cancer cell line panel (NCI60),vero (African green monkey Chlorocebus kidney epithelial cell line)cells, MC3T3 (embryonic calvarium) cells, GH3 (pituitary tumor) cells,PC12 (pheochromocytoma) cells, dog MDCK kidney epithelial cells, XenopusA6 kidney epithelial cells, zebrafish AB9 cells, and Sf9 insectepithelial cells.

Additional examples of adherent cells are shown in Table 1 andcatalogued, for example, in “A Catalog of in Vitro Cell Lines,Transplantable Animal and Human Tumors and Yeast,” The Division ofCancer Treatment and Diagnosis (DCTD), National Cancer Institute (2013),and in Abaan et al., “The exomes of the NCI-60 panel: a genomic resourcefor cancer biology and systems pharmacology,” Cancer Research73(14):4372-82, 2013, the entire contents of each of which areincorporated by reference herein.

TABLE 1 Examples of adherent cells Cell Line Species Organ of OriginDisease BT549 Human Breast Ductal Carcinoma HS 578T Human BreastCarcinoma MCF7 Human Breast Adenocarcinoma MDA-MB-231 Human BreastAdenocarcinoma MDA-MB-468 Human Breast Adenocarcinoma T-47D Human BreastDuctal Carcinoma SF268 Human CNS Anaplastic Astrocytoma SF295 Human CNSGlioblastoma-Multiforme SF539 Human CNS Glioblastoma SNB-19 Human CNSGlioblastoma SNB-75 Human CNS Astrocytoma U251 Human CNS GlioblastomaColo205 Human Colon Dukes’ type D, Colorectal adenocarcinoma HCC 2998Human Colon Carcinoma HCT-116 Human Colon Carcinoma HCT-15 Human ColonDukes’ type C, Colorectal adenocarcinoma HT29 Human Colon Colorectaladenocarcinoma KM12 Human Colon Adenocarcinoma, Grade III SW620 HumanColon Adenocarcinoma 786-O Human Kidney renal cell adenocarcinoma A498Human Kidney Adenocarcinoma ACHN Human Kidney renal cell adenocarcinomaCAKI Human Kidney clear cell carcinoma RXF 393 Human Kidney PoorlyDifferentiated Hypernephroma SN12C Human Kidney Carcinoma TK-10 HumanKidney Spindle Cell carcinoma UO-31 Human Kidney Carcinoma A549 HumanLung Adenocarcinoma EKVX Human Lung Adenocarcinoma HOP-62 Human LungAdenocarcinoma HOP-92 Human Lung Large Cell, Undifferentiated NCI-H226Human Lung squamous cell carcinoma; mesothelioma NCI-H23 Human Lungadenocarcinoma; non-small cell lung cancer NCI-H460 Human Lungcarcinoma; large cell lung cancer NCI-H522 Human Lung adenocarcinoma;non-small cell lung cancer LOX IMVI Human Melanoma Malignant Amelanoticmelanoma M14 Human Melanoma malignant melanoma MALME-3M Human Melanomamalignant melanoma MDA-MB-435 Human Melanoma Adenocarcinoma SK-MEL-2Human Melanoma malignant melanoma SK-MEL-28 Human Melanoma malignantmelanoma SK-MEL-5 Human Melanoma malignant melanoma UACC-257 HumanMelanoma malignant melanoma UACC-62 Human Melanoma malignant melanomaIGROV1 Human Ovary Cystoadenocarcinoma OVCAR-3 Human OvaryAdenocarcinoma OVCAR-4 Human Ovary Adenocarcinoma OVCAR-5 Human OvaryAdenocarcinoma OVCAR-8 Human Ovary Adenocarcinoma SK-OV-3 Human OvaryAdenocarcinoma NCI-ADR-RES Human Ovary Adenocarcinoma DU145 HumanProstate Carcinoma PC-3 Human Prostate grade IV, adenocarcinoma

In some embodiments, the adherent cells are cells that correspond to oneor more of the following cell lines: BT549, HS 578T, MCF7, MDA-MB-231,MDA-MB-468, T-47D, SF268, SF295, SF539, SNB-19, SNB-75, U251, Colo205,HCC 2998, HCT-116, HCT-15, HT29, KM12, SW620, 786-O, A498, ACHN, CAKI,RXF 393, SN12C, TK-10, UO-31, A549, EKVX, HOP-62, HOP-92, NCI-H226,NCI-H23, NCI-H460, NCI-H522, LOX IMVI, M14, MALME-3M, MDA-MB-435, SK-,EL-2, SK-MEL-28, SK-MEL-5, UACC-257, UACC-62, IGROV1, OVCAR-3, OVCAR-4,OVCAR-5, OVCAR-8, SK-OV-3, NCI-ADR-RES, DU145, PC-3, DU145, H295R, HeLa,KBM-7, LNCaP, MCF-7, MDA-MB-468, PC3, SaOS-2, SH-SY5Y, T-47D, THP-1,U87, vero, MC3T3, GH3, PC12, dog MDCK kidney epithelial, Xenopus A6kidney epithelial, zebrafish AB9, and Sf9 insect epithelial cell lines.

Tissue Permeabilization

In some embodiments, a biological sample can be permeabilized tofacilitate transfer of analytes out of the sample, and/or to facilitatetransfer of species (such as capture probes) into the sample. If asample is not permeabilized sufficiently, the amount of analyte capturedfrom the sample may be too low to enable adequate analysis. Conversely,if the tissue sample is too permeable, the relative spatial relationshipof the analytes within the tissue sample can be lost. Hence, a balancebetween permeabilizing the tissue sample enough to obtain good signalintensity while still maintaining the spatial resolution of the analytedistribution in the sample is desirable.

In general, a biological sample can be permeabilized by exposing thesample to one or more permeabilizing agents. Suitable agents for thispurpose include, but are not limited to, organic solvents (e.g.,acetone, ethanol, and methanol), cross-linking agents (e.g.,paraformaldehyde), detergents (e.g., saponin, Triton X-100™ orTween-20™), and enzymes (e.g., trypsin, proteases). In some embodiments,the biological sample can be incubated with a cellular permeabilizingagent to facilitate permeabilization of the sample. Additional methodsfor sample permeabilization are described, for example, in Jamur et al.,Method Mol. Biol. 588:63-66, 2010, the entire contents of which areincorporated herein by reference. Any suitable method for samplepermeabilization can generally be used in connection with the samplesdescribed herein.

In some embodiments, where a diffusion-resistant medium is used to limitmigration of analytes or other species during the analytical procedure,the diffusion-resistant medium can include at least one permeabilizationreagent. For example, the diffusion-resistant medium can include wells(e.g., micro-, nano-, or picowells) containing a permeabilization bufferor reagents. In some embodiments, where the diffusion-resistant mediumis a hydrogel, the hydrogel can include a permeabilization buffer. Insome embodiments, the hydrogel is soaked in permeabilization bufferprior to contacting the hydrogel with a sample. In some embodiments, thehydrogel or other diffusion-resistant medium can contain dried reagentsor monomers to deliver permeabilization reagents when thediffusion-resistant medium is applied to a biological sample. In someembodiments, the diffusion-resistant medium, (i.e. hydrogel) iscovalently attached to a solid substrate (i.e. an acrylated glassslide). In some embodiments, the hydrogel can be modified to bothcontain capture probes and deliver permeabilization reagents. Forexample, a hydrogel film can be modified to include spatially-barcodedcapture probes. The spatially-barcoded hydrogel film is then soaked inpermeabilization buffer before contacting the spatially-barcodedhydrogel film to the sample. The spatially-barcoded hydrogel film thusdelivers permeabilization reagents to a sample surface in contact withthe spatially-barcoded hydrogel, enhancing analyte migration andcapture. In some embodiments, the spatially-barcoded hydrogel is appliedto a sample and placed in a permeabilization bulk solution. In someembodiments, the hydrogel film soaked in permeabilization reagents issandwiched between a sample and a spatially-barcoded array. In someembodiments, target analytes are able to diffuse through thepermeabilizing reagent soaked hydrogel and hybridize or bind the captureprobes on the other side of the hydrogel. In some embodiments, thethickness of the hydrogel is proportional to the resolution loss. Insome embodiments, wells (e.g., micro-, nano-, or picowells) can containspatially-barcoded capture probes and permeabilization reagents and/orbuffer. In some embodiments, spatially-barcoded capture probes andpermeabilization reagents are held between spacers. In some embodiments,the sample is punch, cut, or transferred into the well, wherein a targetanalyte diffuses through the permeabilization reagent/buffer and to thespatially-barcoded capture probes. In some embodiments, resolution lossmay be proportional to gap thickness (e.g. the amount ofpermeabilization buffer between the sample and the capture probes). Insome embodiments, the diffusion-resistant medium (e.g. hydrogel) isbetween approximately 50-500 micrometers thick including 500, 450, 400,350, 300, 250, 200, 150, 100, or 50 micrometers thick, or any thicknesswithin 50 and 500 micrometers.

In some embodiments, permeabilization solution can be delivered to asample through a porous membrane. In some embodiments, a porous membraneis used to limit diffusive analyte losses, while allowingpermeabilization reagents to reach a sample. Membrane chemistry and poresize can be manipulated to minimize analyte loss. In some embodiments,the porous membrane may be made of glass, silicon, paper, hydrogel,polymer monoliths, or other material. In some embodiments, the materialmay be naturally porous. In some embodiments, the material may havepores or wells etched into solid material. In some embodiments, thepermeabilization reagents are flowed through a microfluidic chamber orchannel over the porous membrane. In some embodiments, the flow controlsthe sample’s access to the permeabilization reagents. In someembodiments, a porous membrane is sandwiched between aspatially-barcoded array and the sample, wherein permeabilizationsolution is applied over the porous membrane. The permeabilizationreagents diffuse through the pores of the membrane and into the tissue.

In some embodiments, the biological sample can be permeabilized byadding one or more lysis reagents to the sample. Examples of suitablelysis agents include, but are not limited to, bioactive reagents such aslysis enzymes that are used for lysis of different cell types, e.g.,gram positive or negative bacteria, plants, yeast, mammalian, such aslysozymes, achromopeptidase, lysostaphin, labiase, kitalase, lyticase,and a variety of other commercially available lysis enzymes.

Other lysis agents can additionally or alternatively be added to thebiological sample to facilitate permeabilization. For example,surfactant-based lysis solutions can be used to lyse sample cells. Lysissolutions can include ionic surfactants such as, for example, sarcosyland sodium dodecyl sulfate (SDS). More generally, chemical lysis agentscan include, without limitation, organic solvents, chelating agents,detergents, surfactants, and chaotropic agents.

In some embodiments, the biological sample can be permeabilized bynon-chemical permeabilization methods. Non-chemical permeabilizationmethods are known in the art. For example, non-chemical permeabilizationmethods that can be used include, but are not limited to, physical lysistechniques such as electroporation, mechanical permeabilization methods(e.g., bead beating using a homogenizer and grinding balls tomechanically disrupt sample tissue structures), acousticpermeabilization (e.g., sonication), and thermal lysis techniques suchas heating to induce thermal permeabilization of the sample.

Selective Enrichment of RNA Species

In some embodiments, where RNA is the analyte, one or more RNA analytespecies of interest can be selectively enriched. For example, one ormore species of RNA of interest can be selected by addition of one ormore oligonucleotides to the sample. In some embodiments, the additionaloligonucleotide is a sequence used for priming a reaction by apolymerase. For example, one or more primer sequences with sequencecomplementarity to one or more RNAs of interest can be used to amplifythe one or more RNAs of interest, thereby selectively enriching theseRNAs. In some embodiments, an oligonucleotide with sequencecomplementarity to the complementary strand of captured RNA (e.g., cDNA)can bind to the cDNA. For example, biotinylated oligonucleotides withsequence complementary to one or more cDNA of interest binds to the cDNAand can be selected using biotinylation-strepavidin affinity using anyof a variety of methods known to the field (e.g., streptavidin beads).

Alternatively, one or more species of RNA can be down-selected (e.g.,removed) using any of a variety of methods. For example, probes can beadministered to a sample that selectively hybridize to ribosomal RNA(rRNA), thereby reducing the pool and concentration of rRNA in thesample. Subsequent application of the capture probes to the sample canresult in improved capture of other types of RNA due to the reduction innon-specific RNA present in the sample. Additionally and alternatively,duplex-specific nuclease (DSN) treatment can remove rRNA (see, e.g.,Archer, et al, Selective and flexible depletion of problematic sequencesfrom RNA-seq libraries at the cDNA stage, BMC Genomics, 15 401, (2014),the entire contents of which are incorporated herein by reference).Furthermore, hydroxyapatite chromatography can remove abundant species(e.g., rRNA) (see, e.g., Vandernoot, V.A., cDNA normalization byhydroxyapatite chromatography to enrich transcriptome diversity inRNA-seq applications, Biotechniques, 53(6) 373-80, (2012), the entirecontents of which are incorporated herein by reference).

Other Reagents

Additional reagents can be added to a biological sample to performvarious functions prior to analysis of the sample. In some embodiments,DNase and RNase inactivating agents or inhibitors such as proteinase K,and/or chelating agents such as EDTA, can be added to the sample.

In some embodiments, the sample can be treated with one or more enzymes.For example, one or more endonucleases to fragment DNA, DNA polymeraseenzymes, and dNTPs used to amplify nucleic acids can be added. Otherenzymes that can also be added to the sample include, but are notlimited to, polymerase, transposase, ligase, and DNAse, and RNAse.

In some embodiments, reverse transcriptase enzymes can be added to thesample, including enzymes with terminal transferase activity, primers,and switch oligonucleotides. Template switching can be used to increasethe length of a cDNA, e.g., by appending a predefined nucleic acidsequence to the cDNA.

Pre-Processing for Capture Probe Interaction

In some embodiments, analytes in a biological sample can bepre-processed prior to interaction with a capture probe. For example,prior to interaction with capture probes, polymerization reactionscatalyzed by a polymerase (e.g., DNA polymerase or reversetranscriptase) are performed in the biological sample. In someembodiments, a primer for the polymerization reaction includes afunctional group that enhances hybridization with the capture probe. Thecapture probes can include appropriate capture domains to capturebiological analytes of interest (e.g., poly(dT) sequence to capturepoly(A) mRNA).

In some embodiments, biological analytes are pre-processed for librarygeneration via next generation sequencing. For example, analytes can bepre-processed by addition of a modification (e.g., ligation of sequencesthat allow interaction with capture probes). In some embodiments,analytes (e.g., DNA or RNA) are fragmented using fragmentationtechniques (e.g., using transposases and/or fragmentation buffers).

Fragmentation can be followed by a modification of the analyte. Forexample, a modification can be the addition through ligation of anadapter sequence that allows hybridization with the capture probe. Insome embodiments, where the analyte of interest is RNA, poly(A) tailingis performed. Addition of a poly(A) tail to RNA that does not contain apoly(A) tail can facilitate hybridization with a capture probe thatincludes a capture domain with a functional amount of poly(dT) sequence.

In some embodiments, prior to interaction with capture probes, ligationreactions catalyzed by a ligase are performed in the biological sample.In some embodiments, ligation can be performed by chemical ligation. Insome embodiments, the ligation can be performed using click chemistry asfurther below. In some embodiments, the capture domain includes a DNAsequence that has complementarity to a RNA molecule, where the RNAmolecule has complementarity to a second DNA sequence, and where theRNA-DNA sequence complementarity is used to ligate the second DNAsequence to the DNA sequence in the capture domain. In theseembodiments, direct detection of RNA molecules is possible.

In some embodiments, prior to interaction with capture probes,target-specific reactions are performed in the biological sample.Examples of target specific reactions include, but are not limited to,ligation of target specific adaptors, probes and/or otheroligonucleotides, target specific amplification using primers specificto one or more analytes, and target-specific detection using in situhybridization, DNA microscopy, and/or antibody detection. In someembodiments, a capture probe includes capture domains targeted totarget-specific products (e.g., amplification or ligation).

II. General Spatial Array-Based Analytical Methodology

This section of the disclosure describes methods, apparatus, systems,and compositions for spatial array-based analysis of biological samples.

(A) Spatial Analysis Methods

Array-based spatial analysis methods involve the transfer of one or moreanalytes from a biological sample to an array of features on asubstrate, each of which is associated with a unique spatial location onthe array. Subsequent analysis of the transferred analytes includesdetermining the identity of the analytes and the spatial location ofeach analyte within the sample. The spatial location of each analytewithin the sample is determined based on the feature to which eachanalyte is bound in the array, and the feature’s relative spatiallocation within the array.

There are at least two general methods to associate a spatial barcodewith one or more neighboring cells, such that the spatial barcodeidentifies the one or more cells, and/or contents of the one or morecells, as associated with a particular spatial location. One generalmethod is to drive target analytes out of a cell and towards thespatially-barcoded array. FIG. 1 depicts an exemplary embodiment of thisgeneral method. In FIG. 1 , the spatially-barcoded array populated withcapture probes (as described further herein) is contacted with a sample101, and sample is permeabilized, allowing the target analyte to migrateaway from the sample and toward the array. The target analyte interactswith a capture probe on the spatially-barcoded array 102. Once thetarget analyte hybridizes/is bound to the capture probe, the sample isoptionally removed from the array and the capture probes are analyzed inorder to obtain spatially-resolved analyte information 103.

Another general method is to cleave the spatially-barcoded captureprobes from an array, and drive the spatially-barcoded capture probestowards and/or into or onto the sample. FIG. 2 depicts an exemplaryembodiment of this general method, the spatially-barcoded arraypopulated with capture probes (as described further herein) can becontacted with a sample 201. The spatially-barcoded capture probes arecleaved and then interact with cells within the provided sample 202. Theinteraction can be a covalent or non-covalent cell-surface interaction.The interaction can be an intracellular interaction facilitated by adelivery system or a cell penetration peptide. Once thespatially-barcoded capture probe is associated with a particular cell,the sample can be optionally removed for analysis. The sample can beoptionally dissociated before analysis. Once the tagged cell isassociated with the spatially-barcoded capture probe, the capture probescan be analyzed to obtain spatially-resolved information about thetagged cell 203.

FIG. 3 shows an exemplary workflow that includes preparing a sample on aspatially-barcoded array 301. Sample preparation may include placing thesample on a slide, fixing the sample, and/or staining the sample forimaging. The stained sample is then imaged on the array 302 using bothbrightfield (to image the sample hematoxylin and eosin stain) andfluorescence (to image features) modalities. In some embodiments, targetanalytes are then released from the sample and capture probes formingthe spatially-barcoded array hybridize or bind the released targetanalytes 303. The sample is then removed from the array 304 and thecapture probes cleaved from the array 305. The sample and array are thenoptionally imaged a second time in both modalities 305B while theanalytes are reverse transcribed into cDNA, and an amplicon library isprepared 306 and sequenced 307. The two sets of images are thenspatially-overlaid in order to correlate spatially-identified sampleinformation 308. When the sample and array are not imaged a second time,305B, a spot coordinate file is supplied by the manufacturer instead.The spot coordinate file replaces the second imaging step 305B. Further,amplicon library preparation 306 can be performed with a unique PCRadapter and sequenced 307.

FIG. 4 shows another exemplary workflow that utilizes aspatially-labelled array on a substrate, where capture probes labelledwith spatial barcodes are clustered at areas called features. Thespatially-labelled capture probes can include a cleavage domain, one ormore functional sequences, a spatial barcode, a unique molecularidentifier, and a capture domain. The spatially-labelled capture probescan also include a 5′ end modification for reversible attachment to thesubstrate. The spatially-barcoded array is contacted with a sample 401,and the sample is permeabilized through application of permeabilizationreagents 402. Permeabilization reagents may be administered by placingthe array/sample assembly within a bulk solution. Alternatively,permeabilization reagents may be administered to the sample via adiffusion-resistant medium and/or a physical barrier such as a lid,wherein the sample is sandwiched between the diffusion-resistant mediumand/or barrier and the array-containing substrate. The analytes aremigrated toward the spatially-barcoded capture array using any number oftechniques disclosed herein. For example, analyte migration can occurusing a diffusion-resistant medium lid and passive migration. As anotherexample, analyte migration can be active migration, using anelectrophoretic transfer system, for example. Once the analytes are inclose proximity to the spatially-barcoded capture probes, the captureprobes can hybridize or otherwise bind a target analyte 403. The samplecan be optionally removed from the array 404.

The capture probes can be optionally cleaved from the array 405, and thecaptured analytes can be spatially-tagged by performing a reversetranscriptase first strand cDNA reaction. A first strand cDNA reactioncan be optionally performed using template switching oligonucleotides.For example, a template switching oligonucleotide can hybridize to apoly(C) tail added to a 3′end of the cDNA by a reverse transcriptaseenzyme. The original mRNA template and template switchingoligonucleotide can then be denatured from the cDNA and the barcodedcapture probe can then hybridize with the cDNA and a complement of thecDNA can be generated. The first strand cDNA can then be purified andcollected for downstream amplification steps. The first strand cDNA canbe amplified using PCR 406, wherein the forward and reverse primersflank the spatial barcode and target analyte regions of interest,generating a library associated with a particular spatial barcode. Insome embodiments, the cDNA comprises a sequencing by synthesis (SBS)primer sequence. The library amplicons are sequenced and analyzed todecode spatial information 407.

FIG. 5 depicts an exemplary workflow where the sample is removed fromthe spatially-barcoded array and the spatially-barcoded capture probesare removed from the array for barcoded analyte amplification andlibrary preparation. Another embodiment includes performing first strandsynthesis using template switching oligonucleotides on thespatially-barcoded array without cleaving the capture probes. In thisembodiment, sample preparation 501 and permeabilization 502 areperformed as described elsewhere herein. Once the capture probes capturethe target analyte(s), first strand cDNA created by template switchingand reverse transcriptase 503 is then denatured and the second strand isthen extended 504. The second strand cDNA is then denatured from thefirst strand cDNA, neutralized, and transferred to a tube 505. cDNAquantification and amplification can be performed using standardtechniques discussed herein. The cDNA can then be subjected to librarypreparation 506 and indexing 507, including fragmentation, end-repair,and a-tailing, and indexing PCR steps.

In some non-limiting examples of the workflow above, the sample can beimmersed in 100% chilled methanol and incubated for 30 minutes at -20°C. After 20 minutes, the sample can be removed and rinsed in ultrapurewater. After rinsing the sample, fresh eosin solution is prepared, andthe sample can be covered in isopropanol. After incubating the sample inisopropanol for 1 minute, the reagent can be removed by holding theslide at an angle, where the bottom edge of the slide can be in contactwith a laboratory wipe and air dried. The sample can be uniformlycovered in hematoxylin solution and incubated for 7 minutes at roomtemperature. After incubating the sample in hematoxylin for 7 minutes,the reagent can be removed by holding the slide at an angle, where thebottom edge of the slide can be in contact with a laboratory wipe. Theslide containing the sample can be immersed in water and the excessliquid can be removed. After that, the sample can be covered withblueing buffer and can be incubated for 2 minutes at room temperature.The slide containing the sample can again be immersed in water, anduniformly covered with eosin solution and incubated for 1 minute at roomtemperature. The slide can be air-dried and incubated for 5 minutes at37° C. The sample can be imaged using the methods disclosed herein.

The following are non-limiting, exemplary steps for samplepermeabilization and cDNA generation. The sample can be exposed to apermeabilization enzyme and incubated for 6 minutes at 37° C. Otherpermeabilization methods are described herein. The permeabilizationenzyme can be removed and the sample prepared for analyte capture byadding SSC buffer. The sample can then subjected to a pre-equilibrationthermocycling protocol and the SSC buffer can be removed. A Master Mix,containing nuclease-free water, a reverse transcriptase reagent, atemplate switch oligo, a reducing agent, and a reverse transcriptaseenzyme can be added, and the sample with the Master Mix can be subjectedto a thermocycling protocol. The reagents can be removed from the sampleand NaOH can be applied and incubated for 5 minutes at room temperature.The NaOH can be removed and elution buffer can be added and removed fromthe sample. A Second Strand Mix, including a second strand reagent, asecond strand primer, and a second strand enzyme, can be added to thesample and the sample can be sealed and incubated. At the end of theincubation, the reagents can be removed and elution buffer can be addedand removed from the sample, and NaOH can be added again to the sampleand the sample can be incubated for 10 minutes at room temperature.Tris-HCl can be added and the reagents can be mixed.

The following steps are non-limiting, exemplary steps for cDNAamplification and quality control. A qPCR Mix, including nuclease-freewater, qPCR Master Mix, and cDNA primers, can be prepared and theNaOH/Tris-HCl mix can be mixed with the qPCR Mix and the sample, andthermocycled according to a predetermined thermocycling protocol. Aftercompleting the thermocycling, a cDNA amplification mix can be preparedand combined with the sample and mixed. The sample can then be incubatedand thermocycled. The sample can then be resuspended in SPRIselectReagent and pipetted to ensure proper mixing. The sample can then beincubated at 5 minutes at room temperature, and cleared by placing thesample on a magnet (e.g., the magnet is in the high position). Thesupernatant can be removed and 80% ethanol can be added to the pellet,and incubated for 30 seconds. The ethanol can be removed and the pelletcan be washed again. The sample can then be centrifuged and placed on amagnet (e.g., the magnet is on the low position). Any remaining ethanolcan be removed and the sample can be air dried. The magnet can beremoved and elution buffer can be added to the sample, mixed, andincubated for 2 minutes at room temperature. The sample can then beplaced on the magnet (e.g., on high position) until the solution clears.A portion of the sample can be run on an Agilent Bioanalyzer HighSensitivity chip, where a region can be selected and the cDNAconcentration can be measured to calculate the total cDNA yield.Alternatively, the quantification can be determined by AgilentBioanalyzer or Agilent TapeStation.

The following steps are non-limiting, exemplary steps for spatial geneexpression library construction. A Fragmentation Mix, including afragmentation buffer and fragmentation enzyme, can be prepared on ice.Elution buffer and fragmentation mix can be added to each sample, mixed,and centrifuged. The sample mix can then be placed in a thermocycler andcycled according to a predetermined protocol. The SPRIselect Reagent canbe added to the sample and incubated at 5 minutes at room temperature.The sample can be placed on a magnet (e.g., in the high position) untilthe solution clears, and the supernatant can be transferred to a newtube strip. SPRIselect Reagent can be added to the sample, mixed, andincubated for 5 minutes at room temperature. The sample can be placed ona magnet (e.g., in the high position) until the solution clears. Thesupernatant can be removed and 80% ethanol can be added to the pellet,the pellet can be incubated for 30 seconds, and the ethanol can beremoved. The ethanol wash can be repeated and the sample placed on amagnet (e.g., in the low position) until the solution clears. Theremaining ethanol can be removed and elution buffer can be added to thesample, mixed, and incubated for 2 minutes at room temperature. Thesample can be placed on a magnet (e.g., in the high position) until thesolution clears, and a portion of the sample can be moved to a new tubestrip. An Adaptor Ligation Mix, including ligation buffer, DNA ligase,and adaptor oligos, can be prepared and centrifuged. The AdaptorLigation Mix can be added to the sample, pipette-mixed, and centrifugedbriefly. The sample can then be thermocycled according to apredetermined protocol. The SPRIsleect Reagent can be added to thesample, incubated for 5 minutes at room temperature, and placed on amagnet (e.g.., in the high position) until the solution clears. Thesupernatant can be removed and the pellet can be washed with 80%ethanol, incubated for 30 seconds, and the ethanol can be removed. Theethanol wash can be repeated, and the sample can be centrifuged brieflybefore placing the sample on a magnet (e.g., in the low position). Anyremaining ethanol can be removed and the sample can be air dried.Elution buffer can be added to the sample, the sample can be removedfrom the magnet, and the sample can be pipette-mixed, incubated for 2minutes at room temperature, and placed on a magnet (e.g., in the lowposition) until the solution clears. A portion of the sample can betransferred to a new tube strip. A Sample Index PCR Mix, includingamplification mix and SI primer, can be prepared and combined with thesample. The sample / Sample Index PCR Mix can be loaded into anindividual Chromium i7 Sample Index well and a thermocycling protocolcan be used. SPRIselect Reagent can be added to each sample, mixed, andincubated for 5 minutes at room temperature. The sample can be placed ona magnet (e.g., in the high position) until the solution clears, and thesupernatant can be transferred to a new tube strip. The SPRIselectReagent can be added to each sample, pipette-mixed, and incubated for 5minutes at room temperature. The sample can then be placed on a magnet(e.g., in the high position) until the solution clears. The supernatantcan be removed, and the pellet can be washed with 80% ethanol, incubatedfor 30 seconds, and then the ethanol can be removed. The ethanol washcan be repeated, the sample centrifuged, and placed on a magnet (e.g.,in the low position) to remove any remaining ethanol. The sample can beremoved from the magnet and Elution Buffer can be added to the sample,pipette-mixed, and incubated at 2 minutes at room temperature. Thesample can be placed on a magnet (e.g., in the low position) until thesolution clears and a portion of the sample can be transferred to a newtube strip. The average fragment size can be determined using aBioanalyzer trace or an Agilent TapeStation.

In some embodiments, performing correlative analysis of data produced bythis workflow, and other workflows described herein, can yield over 95%correlation of genes expressed across two capture areas (e.g. 95% orgreater, 96% or greater, 97% or greater, 98% or greater, or 99% orgreater). When performing the described workflows using single cell RNAsequencing of nuclei, in some embodiments, correlative analysis of thedata can yield over 90% (e.g. over 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99%) correlation of genes expressed across two captureareas.

(B) Capture Probes

A “capture probe” refers to any molecule capable of capturing (directlyor indirectly) and/or labelling an analyte of interest in a biologicalsample. In some embodiments, the capture probe is a nucleic acid or apolypeptide. In some embodiments, the capture probe is a conjugate(e.g., an oligonucleotide-antibody conjugate). In some embodiments, thecapture probe includes a barcode (e.g., a spatial barcode and/or aunique molecular identifier (UMI)) and a capture domain.

FIG. 6 is a schematic diagram showing an example of a capture probe, asdescribed herein. As shown, the capture probe 602 is optionally coupledto a feature 601 by a cleavage domain 603, such as a disulfide linker.The capture probe can include functional sequences that are useful forsubsequent processing, such as functional sequence 604, which caninclude a sequencer specific flow cell attachment sequence, e.g., a P5sequence, as well as functional sequence 606, which can includesequencing primer sequences, e.g., a R1 primer binding site. In someembodiments, sequence 604 is a P7 sequence and sequence 606 is a R2primer binding site. A spatial barcode 605 can be included within thecapture probe for use in barcoding the target analyte. The functionalsequences can generally be selected for compatibility with any of avariety of different sequencing systems, e.g., 454 Sequencing, IonTorrent Proton or PGM, Illumina X10, PacBio, Nanopore, etc., and therequirements thereof. In some embodiments, functional sequences can beselected for compatibility with non-commercialized sequencing systems.Examples of such sequencing systems and techniques, for which suitablefunctional sequences can be used, include (but are not limited to) Roche454 sequencing, Ion Torrent Proton or PGM sequencing, Illumina X10sequencing, PacBio SMRT sequencing, and Oxford Nanopore sequencing.Further, in some embodiments, functional sequences can be selected forcompatibility with other sequencing systems, includingnon-commercialized sequencing systems.

In some embodiments, the spatial barcode 605, functional sequences 604(e.g., flow cell attachment sequence) and 606 (e.g., sequencing primersequences) can be common to all of the probes attached to a givenfeature. The spatial barcode can also include a capture domain 607 tofacilitate capture of a target analyte.

Capture Domain

As discussed above, each capture probe includes at least one capturedomain. The “capture domain” is an oligonucleotide, a polypeptide, asmall molecule, or any combination thereof, that binds specifically to adesired analyte. In some embodiments, a capture domain can be used tocapture or detect a desired analyte.

In some embodiments, the capture domain is a functional nucleic acidsequence configured to interact with one or more analytes, such as oneor more different types of nucleic acids (e.g., RNA molecules and DNAmolecules). In some embodiments, the functional nucleic acid sequencecan include an N-mer sequence (e.g., a random N-mer sequence), whichN-mer sequences are configured to interact with a plurality of DNAmolecules. In some embodiments, the functional sequence can include apoly(T) sequence, which poly(T) sequences are configured to interactwith messenger RNA (mRNA) molecules via the poly(A) tail of an mRNAtranscript. In some embodiments, the functional nucleic acid sequence isthe binding target of a protein (e.g., a transcription factor, a DNAbinding protein, or a RNA binding protein), where the analyte ofinterest is a protein.

Capture probes can include ribonucleotides and/or deoxyribonucleotidesas well as synthetic nucleotide residues that are capable ofparticipating in Watson-Crick type or analogous base pair interactions.In some embodiments, the capture domain is capable of priming a reversetranscription reaction to generate cDNA that is complementary to thecaptured RNA molecules. In some embodiments, the capture domain of thecapture probe can prime a DNA extension (polymerase) reaction togenerate DNA that is complementary to the captured DNA molecules. Insome embodiments, the capture domain can template a ligation reactionbetween the captured DNA molecules and a surface probe that is directlyor indirectly immobilized on the substrate. In some embodiments, thecapture domain can be ligated to one strand of the captured DNAmolecules. For example, SplintR ligase along with RNA or DNA sequences(e.g., degenerate RNA) can be used to ligate a single-stranded DNA orRNA to the capture domain. In some embodiments, ligases withRNA-templated ligase activity, e.g., SplintR ligase, T4 RNA ligase 2 orKOD ligase, can be used to ligate a single-stranded DNA or RNA to thecapture domain. In some embodiments, a capture domain includes a splintoligonucleotide. In some embodiments, a capture domain captures a splintoligonucleotide.

In some embodiments, the capture domain is located at the 3′ end of thecapture probe and includes a free 3′ end that can be extended, e.g. bytemplate dependent polymerization, to form an extended capture probe asdescribed herein. In some embodiments, the capture domain includes anucleotide sequence that is capable of hybridizing to nucleic acid, e.g.RNA or other analyte, present in the cells of the tissue samplecontacted with the array. In some embodiments, the capture domain can beselected or designed to bind selectively or specifically to a targetnucleic acid. For example, the capture domain can be selected ordesigned to capture mRNA by way of hybridization to the mRNA poly(A)tail. Thus, in some embodiments, the capture domain includes a poly(T)DNA oligonucleotide, i.e., a series of consecutive deoxythymidineresidues linked by phosphodiester bonds, which is capable of hybridizingto the poly(A) tail of mRNA. In some embodiments, the capture domain caninclude nucleotides that are functionally or structurally analogous to apoly(T) tail. For example, a poly(U) oligonucleotide or anoligonucleotide included of deoxythymidine analogues. In someembodiments, the capture domain includes at least 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20 nucleotides. In some embodiments, the capturedomain includes at least 25, 30, or 35 nucleotides.

In some embodiments, random sequences, e.g., random hexamers or similarsequences, can be used to form all or a part of the capture domain. Forexample, random sequences can be used in conjunction with poly(T) (orpoly(T) analogue) sequences. Thus, where a capture domain includes apoly(T) (or a “poly(T)-like”) oligonucleotide, it can also include arandom oligonucleotide sequence (e.g., “poly(T)-random sequence” probe).This can, for example, be located 5′ or 3′ of the poly(T) sequence, e.g.at the 3′ end of the capture domain. The poly(T)-random sequence probecan facilitate the capture of the mRNA poly(A) tail. In someembodiments, the capture domain can be an entirely random sequence. Insome embodiments, degenerate capture domains can be used.

In some embodiments, a pool of two or more capture probes form amixture, where the capture domain of one or more capture probes includesa poly(T) sequence and the capture domain of one or more capture probesincludes random sequences. In some embodiments, a pool of two or morecapture probes form a mixture where the capture domain of one or morecapture probes includes poly(T)-like sequence and the capture domain ofone or more capture probes includes random sequences. In someembodiments, a pool of two or more capture probes form a mixture wherethe capture domain of one or more capture probes includes apoly(T)-random sequences and the capture domain of one or more captureprobes includes random sequences. In some embodiments, probes withdegenerate capture domains can be added to any of the precedingcombinations listed herein. In some embodiments, probes with degeneratecapture domains can be substituted for one of the probes in each of thepairs described herein.

The capture domain can be based on a particular gene sequence orparticular motif sequence or common/conserved sequence, that it isdesigned to capture (i.e., a sequence-specific capture domain). Thus, insome embodiments, the capture domain is capable of binding selectivelyto a desired sub-type or subset of nucleic acid, for example aparticular type of RNA, such as mRNA, rRNA, tRNA, SRP RNA, tmRNA, snRNA,snoRNA, SmY RNA, scaRNA, gRNA, RNase P, RNase MRP, TERC, SL RNA, aRNA,cis-NAT, crRNA, lncRNA, miRNA, piRNA, siRNA, shRNA, tasiRNA, rasiRNA,7SK, eRNA, ncRNA or other types of RNA. In a non-limiting example, thecapture domain can be capable of binding selectively to a desired subsetof ribonucleic acids, for example, microbiome RNA, such as 16S rRNA.

In some embodiments, a capture domain includes an “anchor” or “anchoringsequence”, which is a sequence of nucleotides that is designed to ensurethat the capture domain hybridizes to the intended biological analyte.In some embodiments, an anchor sequence includes a sequence ofnucleotides, including a 1-mer, 2-mer, 3-mer or longer sequence. In someembodiments, the short sequence is random. For example, a capture domainincluding a poly(T) sequence can be designed to capture an mRNA. In suchembodiments, an anchoring sequence can include a random 3-mer (e.g.,GGG) that helps ensure that the poly(T) capture domain hybridizes to anmRNA. In some embodiments, an anchoring sequence can be VN, N, or NN.Alternatively, the sequence can be designed using a specific sequence ofnucleotides. In some embodiments, the anchor sequence is at the 3′ endof the capture domain. In some embodiments, the anchor sequence is atthe 5′ end of the capture domain.

In some embodiments, capture domains of capture probes are blocked priorto contacting the biological sample with the array, and blocking probesare used when the nucleic acid in the biological sample is modifiedprior to its capture on the array. In some embodiments, the blockingprobe is used to block or modify the free 3′ end of the capture domain.In some embodiments, blocking probes can be hybridized to the captureprobes to mask the free 3′ end of the capture domain, e.g., hairpinprobes or partially double stranded probes. In some embodiments, thefree 3′ end of the capture domain can be blocked by chemicalmodification, e.g., addition of an azidomethyl group as a chemicallyreversible capping moiety such that the capture probes do not include afree 3′ end. Blocking or modifying the capture probes, particularly atthe free 3′ end of the capture domain, prior to contacting thebiological sample with the array, prevents modification of the captureprobes, e.g., prevents the addition of a poly(A) tail to the free 3′ endof the capture probes.

Non-limiting examples of 3′ modifications include dideoxy C-3′ (3′-ddC),3′ inverted dT, 3′ C3 spacer, 3′ Amino, and 3′ phosphorylation. In someembodiments, the nucleic acid in the biological sample can be modifiedsuch that it can be captured by the capture domain. For example, anadaptor sequence (including a binding domain capable of binding to thecapture domain of the capture probe) can be added to the end of thenucleic acid, e.g., fragmented genomic DNA. In some embodiments, this isachieved by ligation of the adaptor sequence or extension of the nucleicacid. In some embodiments, an enzyme is used to incorporate additionalnucleotides at the end of the nucleic acid sequence, e.g., a poly(A)tail. In some embodiments, the capture probes can be reversibly maskedor modified such that the capture domain of the capture probe does notinclude a free 3′ end. In some embodiments, the 3′ end is removed,modified, or made inaccessible so that the capture domain is notsusceptible to the process used to modify the nucleic acid of thebiological sample, e.g., ligation or extension.

In some embodiments, the capture domain of the capture probe is modifiedto allow the removal of any modifications of the capture probe thatoccur during modification of the nucleic acid molecules of thebiological sample. In some embodiments, the capture probes can includean additional sequence downstream of the capture domain, i.e., 3′ to thecapture domain, namely a blocking domain.

In some embodiments, the capture domain of the capture probe can be anon-nucleic acid domain. Examples of suitable capture domains that arenot exclusively nucleic-acid based include, but are not limited to,proteins, peptides, aptamers, antigens, antibodies, and molecularanalogs that mimic the functionality of any of the capture domainsdescribed herein.

Cleavage Domain

Each capture probe can optionally include at least one cleavage domain.The cleavage domain represents the portion of the probe that is used toreversibly attach the probe to an array feature, as will be describedfurther below. Further, one or more segments or regions of the captureprobe can optionally be released from the array feature by cleavage ofthe cleavage domain. As an example spatial barcodes and/or universalmolecular identifiers (UMIs) can be released by cleavage of the cleavagedomain.

FIG. 7 is a schematic illustrating a cleavable capture probe, whereinthe cleaved capture probe can enter into a non-permeabilized cell andbind to target analytes within the sample. The capture probe 701contains a cleavage domain 702, a cell penetrating peptide 703, areporter molecule 704, and a disulfide bond (—S—S—). 705 represents allother parts of a capture probe, for example a spatial barcode and acapture domain.

In some embodiments, the cleavage domain linking the capture probe to afeature is a disulfide bond. A reducing agent can be added to break thedisulfide bonds, resulting in release of the capture probe from thefeature. As another example, heating can also result in degradation ofthe cleavage domain and release of the attached capture probe from thearray feature. In some embodiments, laser radiation is used to heat anddegrade cleavage domains of capture probes at specific locations. Insome embodiments, the cleavage domain is a photo-sensitive chemical bond(i.e., a chemical bond that dissociates when exposed to light such asultraviolet light).

Other examples of cleavage domains include labile chemical bonds suchas, but not limited to, ester linkages (e.g., cleavable with an acid, abase, or hydroxylamine), a vicinal diol linkage (e.g., cleavable viasodium periodate), a Diels-Alder linkage (e.g., cleavable via heat), asulfone linkage (e.g., cleavable via a base), a silyl ether linkage(e.g., cleavable via an acid), a glycosidic linkage (e.g., cleavable viaan amylase), a peptide linkage (e.g., cleavable via a protease), or aphosphodiester linkage (e.g., cleavable via a nuclease (e.g., DNAase)).

In some embodiments, the cleavage domain includes a sequence that isrecognized by one or more enzymes capable of cleaving a nucleic acidmolecule, e.g., capable of breaking the phosphodiester linkage betweentwo or more nucleotides. A bond can be cleavable via other nucleic acidmolecule targeting enzymes, such as restriction enzymes (e.g.,restriction endonucleases). For example, the cleavage domain can includea restriction endonuclease (restriction enzyme) recognition sequence.Restriction enzymes cut double-stranded or single stranded DNA atspecific recognition nucleotide sequences known as restriction sites. Insome embodiments, a rare-cutting restriction enzyme, i.e., enzymes witha long recognition site (at least 8 base pairs in length), is used toreduce the possibility of cleaving elsewhere in the capture probe.

In some embodiments, the cleavage domain includes a poly(U) sequencewhich can be cleaved by a mixture of Uracil DNA glycosylase (UDG) andthe DNA glycosylase-lyase Endonuclease VIII, commercially known as theUSER™ enzyme. Releasable capture probes can be available for reactiononce released. Thus, for example, an activatable capture probe can beactivated by releasing the capture probes from a feature.

In some embodiments, where the capture probe is attached indirectly to asubstrate, e.g., via a surface probe, the cleavage domain includes oneor more mismatch nucleotides, so that the complementary parts of thesurface probe and the capture probe are not 100% complementary (forexample, the number of mismatched base pairs can one, two, or three basepairs). Such a mismatch is recognized, e.g., by the MutY and T7endonuclease I enzymes, which results in cleavage of the nucleic acidmolecule at the position of the mismatch.

In some embodiments, where the capture probe is attached to a featureindirectly, e.g., via a surface probe, the cleavage domain includes anickase recognition site or sequence. Nickases are endonucleases whichcleave only a single strand of a DNA duplex. Thus, the cleavage domaincan include a nickase recognition site close to the 5′ end of thesurface probe (and/or the 5′ end of the capture probe) such thatcleavage of the surface probe or capture probe destabilizes the duplexbetween the surface probe and capture probe thereby releasing thecapture probe) from the feature.

Nickase enzymes can also be used in some embodiments where the captureprobe is attached to the feature directly. For example, the substratecan be contacted with a nucleic acid molecule that hybridizes to thecleavage domain of the capture probe to provide or reconstitute anickase recognition site, e.g., a cleavage helper probe. Thus, contactwith a nickase enzyme will result in cleavage of the cleavage domainthereby releasing the capture probe from the feature. Such cleavagehelper probes can also be used to provide or reconstitute cleavagerecognition sites for other cleavage enzymes, e.g., restriction enzymes.

Some nickases introduce single-stranded nicks only at particular siteson a DNA molecule, by binding to and recognizing a particular nucleotiderecognition sequence. A number of naturally-occurring nickases have beendiscovered, of which at present the sequence recognition properties havebeen determined for at least four. Nickases are described in U.S. Pat.No. 6,867,028, which is incorporated herein by reference in itsentirety. In general, any suitable nickase can be used to bind to acomplementary nickase recognition site of a cleavage domain. Followinguse, the nickase enzyme can be removed from the assay or inactivatedfollowing release of the capture probes to prevent unwanted cleavage ofthe capture probes.

Examples of suitable capture domains that are not exclusivelynucleic-acid based include, but are not limited to, proteins, peptides,aptamers, antigens, antibodies, and molecular analogs that mimic thefunctionality of any of the capture domains described herein.

In some embodiments, a cleavage domain is absent from the capture probe.Examples of substrates with attached capture probes lacking a cleavagedomain are described for example in Macosko et al., (2015) Cell 161,1202-1214, the entire contents of which are incorporated herein byreference.

In some embodiments, the region of the capture probe corresponding tothe cleavage domain can be used for some other function. For example, anadditional region for nucleic acid extension or amplification can beincluded where the cleavage domain would normally be positioned. In suchembodiments, the region can supplement the functional domain or evenexist as an additional functional domain. In some embodiments, thecleavage domain is present but its use is optional.

Functional Domain

Each capture probe can optionally include at least one functionaldomain. Each functional domain typically includes a functionalnucleotide sequence for a downstream analytical step in the overallanalysis procedure.

In some embodiments, the capture probe can include a functional domainfor attachment to a sequencing flow cell, such as, for example, a P5sequence for Illumina® sequencing. In some embodiments, the captureprobe or derivative thereof can include another functional domain, suchas, for example, a P7 sequence for attachment to a sequencing flow cellfor Illumina® sequencing. The functional domains can be selected forcompatibility with a variety of different sequencing systems, e.g., 454Sequencing, Ion Torrent Proton or PGM, Illumina X10, etc., and therequirements thereof.

In some embodiments, the functional domain includes a primer. The primercan include an R1 primer sequence for Illumina® sequencing, and in someembodiments, an R2 primer sequence for Illumina® sequencing. Examples ofsuch capture probes and uses thereof are described in U.S. Pat.Publication Nos. 2014/0378345 and 2015/0376609, the entire contents ofeach of which are incorporated herein by reference.

Spatial Barcode

As discussed above, the capture probe can include one or more spatialbarcodes (e.g., two or more, three or more, four or more, five or more)spatial barcodes. A “spatial barcode” is a contiguous nucleic acidsegment or two or more non-contiguous nucleic acid segments thatfunction as a label or identifier that conveys or is capable ofconveying spatial information. In some embodiments, a capture probeincludes a spatial barcode that possesses a spatial aspect, where thebarcode is associated with a particular location within an array or aparticular location on a substrate.

A spatial barcode can be part of an analyte, or independent from ananalyte (i.e., part of the capture probe). A spatial barcode can be atag attached to an analyte (e.g., a nucleic acid molecule) or acombination of a tag in addition to an endogenous characteristic of theanalyte (e.g., size of the analyte or end sequence(s)). A spatialbarcode can be unique. In some embodiments where the spatial barcode isunique, the spatial barcode functions both as a spatial barcode and as aunique molecular identifier (UMI), associated with one particularcapture probe.

Spatial barcodes can have a variety of different formats. For example,spatial barcodes can include polynucleotide spatial barcodes; randomnucleic acid and/or amino acid sequences; and synthetic nucleic acidand/or amino acid sequences. In some embodiments, a spatial barcode isattached to an analyte in a reversible or irreversible manner. In someembodiments, a spatial barcode is added to, for example, a fragment of aDNA or RNA sample before, during, and/or after sequencing of the sample.In some embodiments, a spatial barcode allows for identification and/orquantification of individual sequencing-reads. In some embodiments, aspatial barcode is a used as a fluorescent barcode for whichfluorescently labeled oligonucleotide probes hybridize to the spatialbarcode.

In some embodiments, the spatial barcode is a nucleic acid sequence thatdoes not substantially hybridize to analyte nucleic acid molecules in abiological sample. In some embodiments, the spatial barcode has lessthan 80% sequence identity (e.g., less than 70%, 60%, 50%, or less than40% sequence identity) to the nucleic acid sequences across asubstantial part (e.g., 80% or more) of the nucleic acid molecules inthe biological sample.

The spatial barcode sequences can include from about 6 to about 20 ormore nucleotides within the sequence of the capture probes. In someembodiments, the length of a spatial barcode sequence can be about 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or longer.In some embodiments, the length of a spatial barcode sequence can be atleast about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20nucleotides or longer. In some embodiments, the length of a spatialbarcode sequence is at most about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20 nucleotides or shorter.

These nucleotides can be completely contiguous, i.e., in a singlestretch of adjacent nucleotides, or they can be separated into two ormore separate subsequences that are separated by 1 or more nucleotides.Separated spatial barcode subsequences can be from about 4 to about 16nucleotides in length. In some embodiments, the spatial barcodesubsequence can be about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16nucleotides or longer. In some embodiments, the spatial barcodesubsequence can be at least about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16 nucleotides or longer. In some embodiments, the spatial barcodesubsequence can be at most about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16 nucleotides or shorter.

For multiple capture probes that are attached to a common array feature,the one or more spatial barcode sequences of the multiple capture probescan include sequences that are the same for all capture probes coupledto the feature, and/or sequences that are different across all captureprobes coupled to the feature.

FIG. 8 is a schematic diagram of an exemplary multiplexedspatially-labelled feature. In FIG. 8 , the feature 801 can be coupledto spatially-barcoded capture probes, wherein the spatially-barcodedprobes of a particular feature can possess the same spatial barcode, buthave different capture domains designed to associate the spatial barcodeof the feature with more than one target analyte. For example, a featuremay be coupled to four different types of spatially-barcoded captureprobes, each type of spatially-barcoded capture probe possessing thespatial barcode 802. One type of capture probe associated with thefeature includes the spatial barcode 802 in combination with a poly(T)capture domain 803, designed to capture mRNA target analytes. A secondtype of capture probe associated with the feature includes the spatialbarcode 802 in combination with a random N-mer capture domain 804 forgDNA analysis. A third type of capture probe associated with the featureincludes the spatial barcode 802 in combination with a capture domaincomplementary to the capture domain on an analyte capture agent captureagent barcode domain 805. A fourth type of capture probe associated withthe feature includes the spatial barcode 802 in combination with acapture probe that can specifically bind a nucleic acid molecule 806that can function in a CRISPR assay (e.g., CRISPR/Cas9). While only fourdifferent capture probe-barcoded constructs are shown in FIG. 8 ,capture-probe barcoded constructs can be tailored for analyses of anygiven analyte associated with a nucleic acid and capable of binding withsuch a construct. For example, the schemes shown in FIG. 8 can also beused for concurrent analysis of other analytes disclosed herein,including, but not limited to: (a) mRNA, a lineage tracing construct,cell surface or intracellular proteins and metabolites, and gDNA; (b)mRNA, accessible chromatin (e.g., ATAC-seq, DNase-seq, and/or MNase-seq)cell surface or intracellular proteins and metabolites, and aperturbation agent (e.g., a CRISPR crRNA/sgRNA, TALEN, zinc fingernuclease, and/or antisense oligonucleotide as described herein); (c)mRNA, cell surface or intracellular proteins and/or metabolites, abarcoded labelling agent (e.g., the MHC multimers described herein), anda V(D)J sequence of an immune cell receptor (e.g., T-cell receptor).

Capture probes attached to a single array feature can include identical(or common) spatial barcode sequences, different spatial barcodesequences, or a combination of both. Capture probes attached to afeature can include multiple sets of capture probes. Capture probes of agiven set can include identical spatial barcode sequences. The identicalspatial barcode sequences can be different from spatial barcodesequences of capture probes of another set.

The plurality of capture probes can include spatial barcode sequences(e.g., nucleic acid barcode sequences) that are associated with specificlocations on a spatial array. For example, a first plurality of captureprobes can be associated with a first region, based on a spatial barcodesequence common to the capture probes within the first region, and asecond plurality of capture probes can be associated with a secondregion, based on a spatial barcode sequence common to the capture probeswithin the second region. The second region may or may not be associatedwith the first region. Additional pluralities of capture probes can beassociated with spatial barcode sequences common to the capture probeswithin other regions. In some embodiments, the spatial barcode sequencescan be the same across a plurality of capture probe molecules.

In some embodiments, multiple different spatial barcodes areincorporated into a single arrayed capture probe. For example, a mixedbut known set of spatial barcode sequences can provide a strongeraddress or attribution of the spatial barcodes to a given spot orlocation, by providing duplicate or independent confirmation of theidentity of the location. In some embodiments, the multiple spatialbarcodes represent increasing specificity of the location of theparticular array point.

Unique Molecular Identifier

The capture probe can include one or more (e.g., two or more, three ormore, four or more, five or more) Unique Molecular Identifiers (UMIs). Aunique molecular identifier is a contiguous nucleic acid segment or twoor more non-contiguous nucleic acid segments that function as a label oridentifier for a particular analyte, or for a capture probe that binds aparticular analyte (e.g., via the capture domain).

A UMI can be unique. A UMI can include one or more specificpolynucleotides sequences, one or more random nucleic acid and/or aminoacid sequences, and/or one or more synthetic nucleic acid and/or aminoacid sequences.

In some embodiments, the UMI is a nucleic acid sequence that does notsubstantially hybridize to analyte nucleic acid molecules in abiological sample. In some embodiments, the UMI has less than 80%sequence identity (e.g., less than 70%, 60%, 50%, or less than 40%sequence identity) to the nucleic acid sequences across a substantialpart (e.g., 80% or more) of the nucleic acid molecules in the biologicalsample.

The UMI can include from about 6 to about 20 or more nucleotides withinthe sequence of the capture probes. In some embodiments, the length of aUMI sequence can be about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20 nucleotides or longer. In some embodiments, the length of aUMI sequence can be at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20 nucleotides or longer. In some embodiments, thelength of a UMI sequence is at most about 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20 nucleotides or shorter.

These nucleotides can be completely contiguous, i.e., in a singlestretch of adjacent nucleotides, or they can be separated into two ormore separate subsequences that are separated by 1 or more nucleotides.Separated UMI subsequences can be from about 4 to about 16 nucleotidesin length. In some embodiments, the UMI subsequence can be about 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or longer. In someembodiments, the UMI subsequence can be at least about 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16 nucleotides or longer. In some embodiments,the UMI subsequence can be at most about 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16 nucleotides or shorter.

In some embodiments, a UMI is attached to an analyte in a reversible orirreversible manner. In some embodiments, a UMI is added to, forexample, a fragment of a DNA or RNA sample before, during, and/or aftersequencing of the analyte. In some embodiments, a UMI allows foridentification and/or quantification of individual sequencing-reads. Insome embodiments, a UMI is a used as a fluorescent barcode for whichfluorescently labeled oligonucleotide probes hybridize to the UMI.

Other Aspects of Capture Probes

For capture probes that are attached to an array feature, an individualarray feature can include one or more capture probes. In someembodiments, an individual array feature includes hundreds or thousandsof capture probes. In some embodiments, the capture probes areassociated with a particular individual feature, where the individualfeature contains a capture probe including a spatial barcode unique to adefined region or location on the array.

In some embodiments, a particular feature can contain capture probesincluding more than one spatial barcode (e.g., one capture probe at aparticular feature can include a spatial barcode that is different thanthe spatial barcode included in another capture probe at the sameparticular feature, while both capture probes include a second, commonspatial barcode), where each spatial barcode corresponds to a particulardefined region or location on the array. For example, multiple spatialbarcode sequences associated with one particular feature on an array canprovide a stronger address or attribution to a given location byproviding duplicate or independent confirmation of the location. In someembodiments, the multiple spatial barcodes represent increasingspecificity of the location of the particular array point. In anon-limiting example, a particular array point can be coded with twodifferent spatial barcodes, where each spatial barcode identifies aparticular defined region within the array, and an array pointpossessing both spatial barcodes identifies the sub-region where twodefined regions overlap, e.g., such as the overlapping portion of a Venndiagram.

In another non-limiting example, a particular array point can be codedwith three different spatial barcodes, where the first spatial barcodeidentifies a first region within the array, the second spatial barcodeidentifies a second region, where the second region is a subregionentirely within the first region, and the third spatial barcodeidentifies a third region, where the third region is a subregionentirely within the first and second subregions.

In some embodiments, capture probes attached to array features arereleased from the array features for sequencing. Alternatively, in someembodiments, capture probes remain attached to the array features, andthe probes are sequenced while remaining attached to the array features(e.g., via in-situ sequencing). Further aspects of the sequencing ofcapture probes are described in subsequent sections of this disclosure.

In some embodiments, an array feature can include different types ofcapture probes attached to the feature. For example, the array featurecan include a first type of capture probe with a capture domain designedto bind to one type of analyte, and a second type of capture probe witha capture domain designed to bind to a second type of analyte. Ingeneral, array features can include one or more (e.g., two or more,three or more, four or more, five or more, six or more, eight or more,ten or more, 12 or more, 15 or more, 20 or more, 30 or more, 50 or more)different types of capture probes attached to a single array feature.

In some embodiments, the capture probe is nucleic acid. In someembodiments, the capture probe is attached to the array feature via its5′ end. In some embodiments, the capture probe includes from the 5′ to3′ end: one or more barcodes (e.g., a spatial barcode and/or a UMI) andone or more capture domains. In some embodiments, the capture probeincludes from the 5′ to 3′ end: one barcode (e.g., a spatial barcode ora UMI) and one capture domain. In some embodiments, the capture probeincludes from the 5′ to 3′ end: a cleavage domain, a functional domain,one or more barcodes (e.g., a spatial barcode and/or a UMI), and acapture domain. In some embodiments, the capture probe includes from the5′ to 3′ end: a cleavage domain, a functional domain, one or morebarcodes (e.g., a spatial barcode and/or a UMI), a second functionaldomain, and a capture domain. In some embodiments, the capture probeincludes from the 5′ to 3′ end: a cleavage domain, a functional domain,a spatial barcode, a UMI, and a capture domain. In some embodiments, thecapture probe does not include a spatial barcode. In some embodiments,the capture probe does not include a UMI. In some embodiments, thecapture probe includes a sequence for initiating a sequencing reaction.

In some embodiments, the capture probe is immobilized on a feature viaits 3′ end. In some embodiments, the capture probe includes from the 3′to 5′ end: one or more barcodes (e.g., a spatial barcode and/or a UMI)and one or more capture domains. In some embodiments, the capture probeincludes from the 3′ to 5′ end: one barcode (e.g., a spatial barcode ora UMI) and one capture domain. In some embodiments, the capture probeincludes from the 3′ to 5′ end: a cleavage domain, a functional domain,one or more barcodes (e.g., a spatial barcode and/or a UMI), and acapture domain. In some embodiments, the capture probe includes from the3′ to 5′ end: a cleavage domain, a functional domain, a spatial barcode,a UMI, and a capture domain.

In some embodiments, a capture probe includes an in situ synthesizedoligonucleotide. In some embodiments, the in situ synthesizedoligonucleotide includes one or more constant sequences, one or more ofwhich serves as a priming sequence (e.g., a primer for amplifying targetnucleic acids). In some embodiments, a constant sequence is a cleavablesequence. In some embodiments, the in situ synthesized oligonucleotideincludes a barcode sequence, e.g., a variable barcode sequence. In someembodiments, the in situ synthesized oligonucleotide is attached to afeature of an array.

In some embodiments, a capture probe is a product of two or moreoligonucleotide sequences, e.g., two or more oligonucleotide sequencesthat are ligated together. In some embodiments, one of theoligonucleotide sequences is an in situ synthesized oligonucleotide.

In some embodiments, the capture probe includes a splintoligonucleotide. Two or more oligonucleotides can be ligated togetherusing a splint oligonucleotide and any variety of ligases known in theart or described herein (e.g., SplintR ligase).

In some embodiments, one of the oligonucleotides includes: a constantsequence (e.g., a sequence complementary to a portion of a splintoligonucleotide), a degenerate sequence, and a capture domain (e.g., asdescribed herein). In some embodiments, the capture probe is generatedby having an enzyme add polynucleotides at the end of an oligonucleotidesequence. The capture probe can include a degenerate sequence, which canfunction as a unique molecular identifier.

A capture probe can include a degenerate sequence, which is a sequencein which some positions of a nucleotide sequence contain a number ofpossible bases. A degenerate sequence can be a degenerate nucleotidesequence including about or at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 nucleotides. In someembodiments, a nucleotide sequence contains 1, 2, 3, 4, 5, 6, 7, 8, 9,0, 10, 15, 20, 25, or more degenerate positions within the nucleotidesequence. In some embodiments, the degenerate sequence is used as a UMI.

In some embodiments, a capture probe includes a restriction endonucleaserecognition sequence or a sequence of nucleotides cleavable by specificenzyme activities. For example, uracil sequences can be cleaved byspecific enzyme activity. As another example, other modified bases(e.g., modified by methylation) can be recognized and cleaved byspecific endonucleases. The capture probes can be subjected to anenzymatic cleavage, which removes the blocking domain and any of theadditional nucleotides that are added to the 3′ end of the capture probeduring the modification process. The removal of the blocking domainreveals and/or restores the free 3′ end of the capture domain of thecapture probe. In some embodiments, additional nucleotides can beremoved to reveal and/or restore the 3′ end of the capture domain of thecapture probe.

In some embodiments, a blocking domain can be incorporated into thecapture probe when it is synthesized, or after its synthesis. Theterminal nucleotide of the capture domain is a reversible terminatornucleotide (e.g., 3′-O-blocked reversible terminator and 3′-unblockedreversible terminator), and can be included in the capture probe duringor after probe synthesis.

Extended Capture Probes

An “extended capture probe” is a capture probe with an enlarged nucleicacid sequence. For example, where the capture probe includes nucleicacid, an “extended 3′ end” indicates that further nucleotides were addedto the most 3′ nucleotide of the capture probe to extend the length ofthe capture probe, for example, by standard polymerization reactionsutilized to extend nucleic acid molecules including templatedpolymerization catalyzed by a polymerase (e.g., a DNA polymerase orreverse transcriptase).

In some embodiments, extending the capture probe includes generatingcDNA from the captured (hybridized) RNA. This process involves synthesisof a complementary strand of the hybridized nucleic acid, e.g.,generating cDNA based on the captured RNA template (the RNA hybridizedto the capture domain of the capture probe). Thus, in an initial step ofextending the capture probe, e.g., the cDNA generation, the captured(hybridized) nucleic acid, e.g., RNA, acts as a template for theextension, e.g., reverse transcription, step.

In some embodiments, the capture probe is extended using reversetranscription. For example, reverse transcription includes synthesizingcDNA (complementary or copy DNA) from RNA, e.g., (messenger RNA), usinga reverse transcriptase. In some embodiments, reverse transcription isperformed while the tissue is still in place, generating an analytelibrary, where the analyte library includes the spatial barcodes fromthe adjacent capture probes. In some embodiments, the capture probe isextended using one or more DNA polymerases.

In some embodiments, the capture domain of the capture probe includes aprimer for producing the complementary strand of the nucleic acidhybridized to the capture probe, e.g., a primer for DNA polymeraseand/or reverse transcription. The nucleic acid, e.g., DNA and/or cDNA,molecules generated by the extension reaction incorporate the sequenceof the capture probe. The extension of the capture probe, e.g., a DNApolymerase and/or reverse transcription reaction, can be performed usinga variety of suitable enzymes and protocols.

In some embodiments, a full-length DNA, e.g. cDNA, molecule isgenerated. In some embodiments, a “full-length” DNA molecule refers tothe whole of the captured nucleic acid molecule. However, if the nucleicacid, e.g. RNA, was partially degraded in the tissue sample, then thecaptured nucleic acid molecules will not be the same length as theinitial RNA in the tissue sample. In some embodiments, the 3′ end of theextended probes, e.g., first strand cDNA molecules, is modified. Forexample, a linker or adaptor can be ligated to the 3′ end of theextended probes. This can be achieved using single stranded ligationenzymes such as T4 RNA ligase or Circligase™ (available from EpicentreBiotechnologies, Madison, WI). In some embodiments, template switchingoligonucleotides are used to extend cDNA in order to generate afull-length cDNA (or as close to a full-length cDNA as possible). Insome embodiments, a second strand synthesis helper probe (a partiallydouble stranded DNA molecule capable of hybridizing to the 3′ end of theextended capture probe), can be ligated to the 3′ end of the extendedprobe, e.g., first strand cDNA, molecule using a double strandedligation enzyme such as T4 DNA ligase. Other enzymes appropriate for theligation step are known in the art and include, e.g., Tth DNA ligase,Taq DNA ligase, Thermococcus sp. (strain 9°N) DNA ligase (9°N™ DNAligase, New England Biolabs), Ampligase™ (available from EpicentreBiotechnologies, Madison, WI), and SplintR (available from New EnglandBiolabs, Ipswich, MA). In some embodiments, a polynucleotide tail, e.g.,a poly(A) tail, is incorporated at the 3′ end of the extended probemolecules. In some embodiments, the polynucleotide tail is incorporatedusing a terminal transferase active enzyme.

In some embodiments, double-stranded extended capture probes are treatedto remove any unextended capture probes prior to amplification and/oranalysis, e.g. sequence analysis. This can be achieved by a variety ofmethods, e.g., using an enzyme to degrade the unextended probes, such asan exonuclease enzyme, or purification columns.

In some embodiments, extended capture probes are amplified to yieldquantities that are sufficient for analysis, e.g., via DNA sequencing.In some embodiments, the first strand of the extended capture probes(e.g., DNA and/or cDNA molecules) acts as a template for theamplification reaction (e.g., a polymerase chain reaction).

In some embodiments, the amplification reaction incorporates an affinitygroup onto the extended capture probe (e.g., RNA-cDNA hybrid) using aprimer including the affinity group. In some embodiments, the primerincludes an affinity group and the extended capture probes includes theaffinity group. The affinity group can correspond to any of the affinitygroups described previously.

In some embodiments, the extended capture probes including the affinitygroup can be coupled to an array feature specific for the affinitygroup. In some embodiments, the substrate can include an antibody orantibody fragment. In some embodiments, the array feature includesavidin or streptavidin and the affinity group includes biotin. In someembodiments, the array feature includes maltose and the affinity groupincludes maltose-binding protein. In some embodiments, the array featureincludes maltose-binding protein and the affinity group includesmaltose. In some embodiments, amplifying the extended capture probes canfunction to release the extended probes from the array feature, insofaras copies of the extended probes are not attached to the array feature.

In some embodiments, the extended capture probe or complement oramplicon thereof is released from an array feature. The step ofreleasing the extended capture probe or complement or amplicon thereoffrom an array feature can be achieved in a number of ways. In someembodiments, an extended capture probe or a complement thereof isreleased from the feature by nucleic acid cleavage and/or bydenaturation (e.g. by heating to denature a double-stranded molecule).

In some embodiments, the extended capture probe or complement oramplicon thereof is released from the array feature by physical means.For example, methods for inducing physical release include denaturingdouble stranded nucleic acid molecules. Another method for releasing theextended capture probes is to use a solution that interferes with thehydrogen bonds of the double stranded molecules. In some embodiments,the extended capture probe is released by applying heated water such aswater or buffer of at least 85° C., e.g., at least 90, 91, 92, 93, 94,95, 96, 97, 98, or 99° C. In some embodiments, a solution includingsalts, surfactants, etc. that can further destabilize the interactionbetween the nucleic acid molecules is added to release the extendedcapture probe from the array feature. In some embodiments, a formamidesolution can be used to destabilize the interaction between nucleic acidmolecules to release the extended capture probe from the array feature.

Analyte Capture Agents

This disclosure also provides methods and materials for using analytecapture agents for spatial profiling of biological analytes (e.g., mRNA,genomic DNA, accessible chromatin, and cell surface or intracellularproteins and/or metabolites). As used herein, an “analyte capture agent”(also referred to previously at times as a “cell labelling agent”)refers to an agent that interacts with an analyte (e.g., an analyte in asample) and with a capture probe (e.g., a capture probe attached to asubstrate) to identify the analyte. In some embodiments, the analytecapture agent includes an analyte binding moiety and a capture agentbarcode domain.

FIG. 9 is a schematic diagram of an exemplary analyte capture agent 902comprised of an analyte binding moiety 904 and a capture agent barcodedomain 908. An analyte binding moiety 904 is a molecule capable ofbinding to an analyte 906 and interacting with a spatially-barcodedcapture probe. The analyte binding moiety can bind to the analyte 906with high affinity and/or with high specificity. The analyte captureagent can include a capture agent barcode domain 908, a nucleotidesequence (e.g., an oligonucleotide), which can hybridize to at least aportion or an entirety of a capture domain of a capture probe. Theanalyte binding moiety 904 can include a polypeptide and/or an aptamer(e.g., an oligonucleotide or peptide molecule that binds to a specifictarget analyte). The analyte binding moiety 904 can include an antibodyor antibody fragment (e.g., an antigen-binding fragment).

As used herein, the term “analyte binding moiety” refers to a moleculeor moiety capable of binding to a macromolecular constituent (e.g., ananalyte, e.g., a biological analyte). In some embodiments of any of thespatial profiling methods described herein, the analyte binding moietyof the analyte capture agent that binds to a biological analyte caninclude, but is not limited to, an antibody, or an epitope bindingfragment thereof, a cell surface receptor binding molecule, a receptorligand, a small molecule, a bi-specific antibody, a bi-specific T-cellengager, a T-cell receptor engager, a B-cell receptor engager, apro-body, an aptamer, a monobody, an affimer, a darpin, and a proteinscaffold, or any combination thereof. The analyte binding moiety canbind to the macromolecular constituent (e.g., analyte) with highaffinity and/or with high specificity. The analyte binding moiety caninclude a nucleotide sequence (e.g., an oligonucleotide), which cancorrespond to at least a portion or an entirety of the analyte bindingmoiety. The analyte binding moiety can include a polypeptide and/or anaptamer (e.g., a polypeptide and/or an aptamer that binds to a specifictarget molecule, e.g., an analyte). The analyte binding moiety caninclude an antibody or antibody fragment (e.g., an antigen-bindingfragment) that binds to a specific analyte (e.g., a polypeptide).

In some embodiments, analyte capture agents are capable of binding toanalytes present inside a cell. In some embodiments, analyte captureagents are capable of binding to cell surface analytes that can include,without limitation, a receptor, an antigen, a surface protein, atransmembrane protein, a cluster of differentiation protein, a proteinchannel, a protein pump, a carrier protein, a phospholipid, aglycoprotein, a glycolipid, a cell-cell interaction protein complex, anantigen-presenting complex, a major histocompatibility complex, anengineered T-cell receptor, a T-cell receptor, a B-cell receptor, achimeric antigen receptor, an extracellular matrix protein, aposttranslational modification (e.g., phosphorylation, glycosylation,ubiquitination, nitrosylation, methylation, acetylation or lipidation)state of a cell surface protein, a gap junction, and an adherensjunction. In some embodiments, the analyte capture agents are capable ofbinding to cell surface analytes that are post-translationally modified.In such embodiments, analyte capture agents can be specific for cellsurface analytes based on a given state of posttranslationalmodification (e.g., phosphorylation, glycosylation, ubiquitination,nitrosylation, methylation, acetylation or lipidation), such that a cellsurface analyte profile can include posttranslational modificationinformation of one or more analytes.

In some embodiments, the analyte capture agent includes a capture agentbarcode domain that is conjugated or otherwise attached to the analytebinding moiety. In some embodiments, the capture agent barcode domain iscovalently-linked to the analyte binding moiety. In some embodiments, acapture agent barcode domain is a nucleic acid sequence. In someembodiments, a capture agent barcode domain includes an analyte bindingmoiety barcode and an analyte capture sequence.

As used herein, the term “analyte binding moiety barcode” refers to abarcode that is associated with or otherwise identifies the analytebinding moiety. In some embodiments, by identifying an analyte bindingmoiety by identifying its associated analyte binding moiety barcode, theanalyte to which the analyte binding moiety binds can also beidentified. An analyte binding moiety barcode can be a nucleic acidsequence of a given length and/or sequence that is associated with theanalyte binding moiety. An analyte binding moiety barcode can generallyinclude any of the variety of aspects of barcodes described herein. Forexample, an analyte capture agent that is specific to one type ofanalyte can have coupled thereto a first capture agent barcode domain(e.g., that includes a first analyte binding moiety barcode), while ananalyte capture agent that is specific to a different analyte can have adifferent capture agent barcode domain (e.g., that includes a secondbarcode analyte binding moiety barcode) coupled thereto. In someaspects, such a capture agent barcode domain can include an analytebinding moiety barcode that permits identification of the analytebinding moiety to which the capture agent barcode domain is coupled. Theselection of the capture agent barcode domain can allow significantdiversity in terms of sequence, while also being readily attachable tomost analyte binding moieties (e.g., antibodies) as well as beingreadily detected, (e.g., using sequencing or array technologies). Insome embodiments, the analyte capture agents can include analyte bindingmoieties with capture agent barcode domains attached to them. Forexample, an analyte capture agent can include a first analyte bindingmoiety (e.g., an antibody that binds to an analyte, e.g., a first cellsurface feature) having associated with it a capture agent barcodedomain that includes a first analyte binding moiety barcode.

In some embodiments, the capture agent barcode domain of an analytecapture agent includes an analyte capture sequence. As used herein, theterm “analyte capture sequence” refers to region or moiety of configuredto hybridize to, bind to, couple to, or otherwise interact with acapture domain of a capture probe. In some embodiments, an analytecapture sequence includes a nucleic acid sequence that is complementaryto or substantially complementary to the capture domain of a captureprobe such that the analyte capture sequence hybridizes to the capturedomain of the capture probe. In some embodiments, an analyte capturesequence comprises a poly(A) nucleic acid sequence that hybridizes to acapture domain that comprises a poly(T) nucleic acid sequence. In someembodiments, an analyte capture sequence comprises a poly(T) nucleicacid sequence that hybridizes to a capture domain that comprises apoly(A) nucleic acid sequence. In some embodiments, an analyte capturesequence comprises a non-homopolymeric nucleic acid sequence thathybridizes to a capture domain that comprises a non-homopolymericnucleic acid sequence that is complementary (or substantiallycomplementary) to the non-homopolymeric nucleic acid sequence of theanalyte capture region.

In some embodiments of any of the spatial analysis methods describedherein that employ an analyte capture agent, the capture agent barcodedomain can be directly coupled to the analyte binding moiety, or theycan be attached to a bead, molecular lattice, e.g., a linear, globular,cross-slinked, or other polymer, or other framework that is attached orotherwise associated with the analyte binding moiety, which allowsattachment of multiple capture agent barcode domains to a single analytebinding moiety. Attachment (coupling) of the capture agent barcodedomains to the analyte binding moieties can be achieved through any of avariety of direct or indirect, covalent or non-covalent associations orattachments. For example, in the case of a capture agent barcode domaincoupled to an analyte binding moiety that includes an antibody orantigen-binding fragment, such capture agent barcode domains can becovalently attached to a portion of the antibody or antigen-bindingfragment using chemical conjugation techniques (e.g., Lightning-Link®antibody labelling kits available from Innova Biosciences). In someembodiments, a capture agent barcode domain can be coupled to anantibody or antigen-binding fragment using non-covalent attachmentmechanisms (e.g., using biotinylated antibodies and oligonucleotides orbeads that include one or more biotinylated linker, coupled tooligonucleotides with an avidin or streptavidin linker.) Antibody andoligonucleotide biotinylation techniques can be used, and are describedfor example in Fang et al., Nucleic Acids Res. (2003), 31(2): 708-715,the entire contents of which are incorporated by reference herein.Likewise, protein and peptide biotinylation techniques have beendeveloped and can be used, and are described for example in U.S. Pat.No. 6,265,552, the entire contents of which are incorporated byreference herein. Furthermore, click reaction chemistry such as amethyltetrazine-PEG5-NHS ester reaction, a TCO-PEG4-NHS ester reaction,or the like, can be used to couple capture agent barcode domains toanalyte binding moieties. The reactive moiety on the analyte bindingmoiety can also include amine for targeting aldehydes, amine fortargeting maleimide (e.g., free thiols), azide for targeting clickchemistry compounds (e.g., alkynes), biotin for targeting streptavidin,phosphates for targeting EDC, which in turn targets active ester (e.g.,NH2). The reactive moiety on the analyte binding moiety can be achemical compound or group that binds to the reactive moiety on theanalyte binding moiety. Exemplary strategies to conjugate the analytebinding moiety to the capture agent barcode domain include the use ofcommercial kits (e.g., Solulink, Thunder link), conjugation of mildreduction of hinge region and maleimide labelling, stain-promoted clickchemistry reaction to labeled amides (e.g., copper-free), andconjugation of periodate oxidation of sugar chain and amine conjugation.In the cases where the analyte binding moiety is an antibody, theantibody can be modified prior to or contemporaneously with conjugationof the oligonucleotide. For example, the antibody can be glycosylatedwith a substrate-permissive mutant of β-1,4-galactosyltransferase, GalT(Y289L) and azide-bearing uridine diphosphate-N-acetylgalactosamineanalog uridine diphosphate -GalNAz. The modified antibody can beconjugated to an oligonucleotide with a dibenzocyclooctyne-PEG4-NHSgroup. In some embodiments, certain steps (e.g., COOH activation (e.g.,EDC) and homobifunctional cross linkers) can be avoided to prevent theanalyte binding moieties from conjugating to themselves. In someembodiments of any of the spatial profiling methods described herein,the analyte capture agent (e.g., analyte binding moiety coupled to anoligonucleotide) can be delivered into the cell, e.g., by transfection(e.g., using transfectamine, cationic polymers, calcium phosphate orelectroporation), by transduction (e.g., using a bacteriophage orrecombinant viral vector), by mechanical delivery (e.g., magneticbeads), by lipid (e.g., 1,2-Dioleoyl-sn-glycero-3-phosphocholine(DOPC)), or by transporter proteins. An analyte capture agent can bedelivered into a cell using exosomes. For example, a first cell can begenerated that releases exosomes comprising an analyte capture agent. Ananalyte capture agent can be attached to an exosome membrane. An analytecapture agent can be contained within the cytosol of an exosome.Released exosomes can be harvested and provided to a second cell,thereby delivering the analyte capture agent into the second cell. Ananalyte capture agent can be releasable from an exosome membrane before,during, or after delivery into a cell. In some embodiments, the cell ispermeabilized to allow the analyte capture agent to couple withintracellular cellular constituents (such as, without limitation,intracellular proteins, metabolites and nuclear membrane proteins).Following intracellular delivery, analyte capture agents can be used toanalyze intracellular constituents as described herein.

In some embodiments of any of the spatial profiling methods describedherein, the capture agent barcode domain coupled to an analyte captureagent can include modifications that render it non-extendable by apolymerase. In some embodiments, when binding to a capture domain of acapture probe or nucleic acid in a sample for a primer extensionreaction, the capture agent barcode domain can serve as a template, nota primer. When the capture agent barcode domain also includes a barcode(e.g., an analyte binding moiety barcode), such a design can increasethe efficiency of molecular barcoding by increasing the affinity betweenthe capture agent barcode domain and unbarcoded sample nucleic acids,and eliminate the potential formation of adaptor artifacts. In someembodiments, the capture agent barcode domain can include a random N-mersequence that is capped with modifications that render it non-extendableby a polymerase. In some cases, the composition of the random N-mersequence can be designed to maximize the binding efficiency to free,unbarcoded ssDNA molecules. The design can include a random sequencecomposition with a higher GC content, a partial random sequence withfixed G or C at specific positions, the use of guanosines, the use oflocked nucleic acids, or any combination thereof.

A modification for blocking primer extension by a polymerase can be acarbon spacer group of different lengths or a dideoxynucleotide. In someembodiments, the modification can be an abasic site that has an apurineor apyrimidine structure, a base analog, or an analogue of a phosphatebackbone, such as a backbone of N-(2-aminoethyl)-glycine linked by amidebonds, tetrahydrofuran, or 1′, 2′-Dideoxyribose. The modification canalso be a uracil base, 2′OMe modified RNA, C3-18 spacers (e.g.,structures with 3-18 consecutive carbon atoms, such as C3 spacer),ethylene glycol multimer spacers (e.g., spacer 18 (hexa-ethyleneglycolspacer), biotin, di-deoxynucleotide triphosphate, ethylene glycol,amine, or phosphate.

In some embodiments of any of the spatial profiling methods describedherein, the capture agent barcode domain coupled to the analyte bindingmoiety includes a cleavable domain. For example, after the analytecapture agent binds to an analyte (e.g., a cell surface analyte), thecapture agent barcode domain can be cleaved and collected for downstreamanalysis according to the methods as described herein. In someembodiments, the cleavable domain of the capture agent barcode domainincludes a U-excising element that allows the species to release fromthe bead. In some embodiments, the U-excising element can include asingle-stranded DNA (ssDNA) sequence that contains at least one uracil.The species can be attached to a bead via the ssDNA sequence. Thespecies can be released by a combination of uracil-DNA glycosylase(e.g., to remove the uracil) and an endonuclease (e.g., to induce anssDNA break). If the endonuclease generates a 5′ phosphate group fromthe cleavage, then additional enzyme treatment can be included indownstream processing to eliminate the phosphate group, e.g., prior toligation of additional sequencing handle elements, e.g., Illumina fullP5 sequence, partial P5 sequence, full R1 sequence, and/or partial R1sequence.

In some embodiments, an analyte binding moiety of an analyte captureagent includes one or more antibodies or antigen binding fragmentsthereof. The antibodies or antigen binding fragments including theanalyte binding moiety can specifically bind to a target analyte. Insome embodiments, the analyte is a protein (e.g., a protein on a surfaceof the biological sample (e.g., a cell) or an intracellular protein). Insome embodiments, a plurality of analyte capture agents comprising aplurality of analyte binding moieties bind a plurality of analytespresent in a biological sample. In some embodiments, the plurality ofanalytes includes a single species of analyte (e.g., a single species ofpolypeptide). In some embodiments in which the plurality of analytesincludes a single species of analyte, the analyte binding moieties ofthe plurality of analyte capture agents are the same. In someembodiments in which the plurality of analytes includes a single speciesof analyte, the analyte binding moieties of the plurality of analytecapture agents are the different (e.g., members of the plurality ofanalyte capture agents can have two or more species of analyte bindingmoieties, wherein each of the two or more species of analyte bindingmoieties binds a single species of analyte, e.g., at different bindingsites). In some embodiments, the plurality of analytes includes multipledifferent species of analyte (e.g., multiple different species ofpolypeptides).

In some embodiments, multiple different species of analytes (e.g.,polypeptides) from the biological sample can be subsequently associatedwith the one or more physical properties of the biological sample. Forexample, the multiple different species of analytes can be associatedwith locations of the analytes in the biological sample. Suchinformation (e.g., proteomic information when the analyte bindingmoiety(ies) recognizes a polypeptide(s)) can be used in association withother spatial information (e.g., genetic information from the biologicalsample, such as DNA sequence information, transcriptome information(i.e., sequences of transcripts), or both). For example, a cell surfaceprotein of a cell can be associated with one or more physical propertiesof the cell (e.g., a shape, size, activity, or a type of the cell). Theone or more physical properties can be characterized by imaging thecell. The cell can be bound by an analyte capture agent comprising ananalyte binding moiety that binds to the cell surface protein and ananalyte binding moiety barcode that identifies that analyte bindingmoiety, and the cell can be subjected to spatial analysis (e.g., any ofthe variety of spatial analysis methods described herein). For example,the analyte capture agent bound to the cell surface protein can be boundto a capture probe (e.g., a capture probe on an array), which captureprobe includes a capture domain that interacts with an analyte capturesequence present on the capture agent barcode domain of the analytecapture agent. All or part of the capture agent barcode domain(including the analyte binding moiety barcode) can be copied with apolymerase using a 3′ end of the capture domain as a priming site,generating an extended capture probe that includes the all or part ofthe capture probe (including a spatial barcode present on the captureprobe) and a copy of the analyte binding moiety barcode. In someembodiments, the spatial array with the extended capture probe(s) can becontacted with a sample, where the analyte capture agent(s) associatedwith the spatial array capture the target analyte(s). The analytecapture agent(s) containing the extended capture probe(s), whichincludes the spatial barcode(s) of the capture probe(s) and the analytebinding moiety barcode(s), can then be denatured from the captureprobe(s) of the spatial array. This allows the spatial array to bereused. The sample can be dissociated into non-aggregated cells (e.g.single cells) and analyzed by the single cell / droplet methodsdescribed herein. The extended capture probe can be sequenced to obtaina nucleic acid sequence, in which the spatial barcode of the captureprobe is associated with the analyte binding moiety barcode of theanalyte capture agent. The nucleic acid sequence of the extended captureprobe can thus be associated with the analyte (e.g., cell surfaceprotein), and in turn, with the one or more physical properties of thecell (e.g., a shape or cell type). In some embodiments, the nucleic acidsequence of the extended capture probe can be associated with anintracellular analyte of a nearby cell, where the intracellular analytewas released using any of the cell permeabilization or analyte migrationtechniques described herein.

In some embodiments of any of the spatial profiling methods describedherein, the capture agent barcode domains released from the analytecapture agents can then be subjected to sequence analysis to identifywhich analyte capture agents were bound to analytes. Based upon thecapture agent barcode domains that are associated with a feature (e.g.,a feature at a particular location) on a spatial array and the presenceof the analyte binding moiety barcode sequence, an analyte profile canbe created for a biological sample. Profiles of individual cells orpopulations of cells can be compared to profiles from other cells, e.g.,‘normal’ cells, to identify variations in analytes, which can providediagnostically relevant information. In some embodiments, these profilescan be useful in the diagnosis of a variety of disorders that arecharacterized by variations in cell surface receptors, such as cancerand other disorders.

FIG. 10 is a schematic diagram depicting an exemplary interactionbetween a feature-immobilized capture probe 1024 and an analyte captureagent 1026. The feature-immobilized capture probe 1024 can include aspatial barcode 1008 as well as one or more functional sequences 1006and 1010, as described elsewhere herein. The capture probe can alsoinclude a capture domain 1012 that is capable of binding to an analytecapture agent 1026. The analyte capture agent 1026 can include afunctional sequence 1018, capture agent barcode domain 1016, and ananalyte capture sequence 1014 that is capable of binding to the capturedomain 1012 of the capture probe 1024. The analyte capture agent canalso include a linker 1020 that allows the capture agent barcode domain1016 to couple to the analyte binding moiety 1022.

In some embodiments of any of the spatial profiling methods describedherein, the methods are used to identify immune cell profiles. Immunecells express various adaptive immunological receptors relating toimmune function, such as T cell receptors (TCRs) and B cell receptors(BCRs). T cell receptors and B cell receptors play a part in the immuneresponse by specifically recognizing and binding to antigens and aidingin their destruction.

The T cell receptor, or TCR, is a molecule found on the surface of Tcells that is generally responsible for recognizing fragments of antigenas peptides bound to major histocompatibility complex (MHC) molecules.The TCR is generally a heterodimer of two chains, each of which is amember of the immunoglobulin superfamily, possessing an N-terminalvariable (V) domain, and a C terminal constant domain. In humans, in 95%of T cells, the TCR consists of an alpha (α) and beta (β) chain, whereasin 5% of T cells, the TCR consists of gamma and delta (γ/δ) chains. Thisratio can change during ontogeny and in diseased states as well as indifferent species. When the TCR engages with antigenic peptide and MHC(peptide/MHC or pMHC), the T lymphocyte is activated through signaltransduction.

Each of the two chains of a TCR contains multiple copies of genesegments - a variable ‘V’ gene segment, a diversity ‘D’ gene segment,and a joining ‘J’ gene segment. The TCR alpha chain (TCRa) is generatedby recombination of V and J segments, while the beta chain (TCRb) isgenerated by recombination of V, D, and J segments. Similarly,generation of the TCR gamma chain involves recombination of V and J genesegments, while generation of the TCR delta chain occurs byrecombination of V, D, and J gene segments. The intersection of thesespecific regions (V and J for the alpha or gamma chain, or V, D and Jfor the beta or delta chain) corresponds to the CDR3 region that isimportant for antigen-MHC recognition. Complementarity determiningregions (e.g., CDR1, CDR2, and CDR3), or hypervariable regions, aresequences in the variable domains of antigen receptors (e.g., T cellreceptor and immunoglobulin) that can complement an antigen. Most of thediversity of CDRs is found in CDR3, with the diversity being generatedby somatic recombination events during the development of T lymphocytes.A unique nucleotide sequence that arises during the gene arrangementprocess can be referred to as a clonotype.

The B cell receptor, or BCR, is a molecule found on the surface of Bcells. The antigen binding portion of a BCR is composed of amembrane-bound antibody that, like most antibodies (e.g.,immunoglobulins), has a unique and randomly determined antigen-bindingsite. The antigen binding portion of a BCR includes membrane-boundimmunoglobulin molecule of one isotype (e.g., IgD, IgM, IgA, IgG, orIgE). When a B cell is activated by its first encounter with a cognateantigen, the cell proliferates and differentiates to generate apopulation of antibody-secreting plasma B cells and memory B cells. Thevarious immunoglobulin isotypes differ in their biological features,structure, target specificity and distribution. A variety of molecularmechanisms exist to generate initial diversity, including geneticrecombination at multiple sites.

The BCR is composed of two genes IgH and IgK (or IgL) coding forantibody heavy and light chains. Immunoglobulins are formed byrecombination among gene segments, sequence diversification at thejunctions of these segments, and point mutations throughout the gene.Each heavy chain gene contains multiple copies of three different genesegments - a variable ‘V’ gene segment, a diversity ‘D’ gene segment,and a joining ‘J’ gene segment. Each light chain gene contains multiplecopies of two different gene segments for the variable region of theprotein - a variable ‘V’ gene segment and a joining ‘J’ gene segment.

The recombination can generate a molecule with one of each of the V, D,and J segments. Furthermore, several bases can be deleted and othersadded (called N and P nucleotides) at each of the two junctions, therebygenerating further diversity. After B cell activation, a process ofaffinity maturation through somatic hypermutation occurs. In thisprocess, progeny cells of the activated B cells accumulate distinctsomatic mutations throughout the gene with higher mutation concentrationin the CDR regions leading to the generation of antibodies with higheraffinity to the antigens.

In addition to somatic hypermutation, activated B cells undergo theprocess of isotype switching. Antibodies with the same variable segmentscan have different forms (isotypes) depending on the constant segment.Whereas all naïve B cells express IgM (or IgD), activated B cells mostlyexpress IgG but also IgM, IgA and IgE. This expression switching fromIgM (and/or IgD) to IgG, IgA, or IgE occurs through a recombinationevent causing one cell to specialize in producing a specific isotype. Aunique nucleotide sequence that arises during the gene arrangementprocess can similarly be referred to as a clonotype.

Certain methods described herein are utilized to analyze the varioussequences of TCRs and BCRs from immune cells, for example, variousclonotypes. In some embodiments, the methods are used to analyze thesequence of a TCR alpha chain, a TCR beta chain, a TCR delta chain, aTCR gamma chain, or any fragment thereof (e.g., variable regionsincluding V(D)J or VJ regions, constant regions, transmembrane regions,fragments thereof, combinations thereof, and combinations of fragmentsthereof). In some embodiments, the methods described herein can be usedto analyze the sequence of a B cell receptor heavy chain, B cellreceptor light chain, or any fragment thereof (e.g., variable regionsincluding V(D)J or VJ regions, constant regions, transmembrane regions,fragments thereof, combinations thereof, and combinations of fragmentsthereof).

Where immune cells are to be analyzed, primer sequences useful in any ofthe various operations for attaching barcode sequences and/oramplification reactions can include gene specific sequences which targetgenes or regions of genes of immune cell proteins, for example immunereceptors. Such gene sequences include, but are not limited to,sequences of various T cell receptor alpha variable genes (TRAV genes),T cell receptor alpha joining genes (TRAJ genes), T cell receptor alphaconstant genes (TRAC genes), T cell receptor beta variable genes (TRBVgenes), T cell receptor beta diversity genes (TRBD genes), T cellreceptor beta joining genes (TRBJ genes), T cell receptor beta constantgenes (TRBC genes), T cell receptor gamma variable genes (TRGV genes), Tcell receptor gamma joining genes (TRGJ genes), T cell receptor gammaconstant genes (TRGC genes), T cell receptor delta variable genes (TRDVgenes), T cell receptor delta diversity genes (TRDD genes), T cellreceptor delta joining genes (TRDJ genes), and T cell receptor deltaconstant genes (TRDC genes).

In some embodiments, the analyte binding moiety is based on the MajorHistocompatibility Complex (MHC) class I or class II. In someembodiments, the analyte binding moiety is an MHC multimer including,without limitation, MHC dextramers, MHC tetramers, and MHC pentamers(see, for example, U.S. Pat. Application Publication Nos. US2018/0180601 and US 2017/0343545, the entire contents of each of whichare incorporated herein by reference. MHCs (e.g., a soluble MHC monomermolecule), including full or partial MHC-peptides, can be used asanalyte binding moieties of analyte capture agents that are coupled tocapture agent barcode domains that include an analyte binding moietybarcode that identifies its associated MHC (and, thus, for example, theMHC’s TCR binding partner). In some embodiments, MHCs are used toanalyze one or more cell-surface features of a T-cell, such as a TCR. Insome cases, multiple MHCs are associated together in a larger complex(MHC multimer) to improve binding affinity of MHCs to TCRs via multipleligand binding synergies. FIGS. 11A, 11B, and 11C are schematicsillustrating how streptavidin cell tags can be utilized in anarray-based system to produce a spatially-barcoded cell or cellularcontents. For example, as shown in FIG. 11 , peptide-bound majorhistocompatibility complex (pMHCs) can be individually associated withbiotin and bound to a streptavidin moiety such that the streptavidinmoiety comprises multiple pMHC moieties. Each of these moieties can bindto a TCR such that the streptavidin binds to a target T-cell viamultiple MCH/TCR binding interactions. Multiple interactions synergizeand can substantially improve binding affinity. Such improved affinitycan improve labelling of T-cells and also reduce the likelihood thatlabels will dissociate from T-cell surfaces. As shown in FIG. 11B, acapture agent barcode domain 1101 can be modified with streptavidin 1102and contacted with multiple molecules of biotinylated MHC 1103 (such asa pMHC) such that the biotinylated MHC 1103 molecules are coupled withthe streptavidin conjugated capture agent barcode domain 1101. Theresult is a barcoded MHC multimer complex 1105. As shown in FIG. 11B,the capture agent barcode domain sequence 1101 can identify the MHC asits associated label and also includes optional functional sequencessuch as sequences for hybridization with other oligonucleotides. Asshown in FIG. 11C, one example oligonucleotide is capture probe 1106that comprises a complementary sequence (e.g., rGrGrG corresponding to CC C), a barcode sequence and other functional sequences, such as, forexample, a UMI, an adapter sequence (e.g., comprising a sequencingprimer sequence (e.g., R1 or a partial R1 (“pR1”)), a flow cellattachment sequence (e.g., P5 or P7 or partial sequences thereof)), etc.In some cases, capture probe 1106 may at first be associated with afeature (e.g., a gel bead) and released from the feature. In otherembodiments, capture probe 1106 can hybridize with a capture agentbarcode domain 1101 of the MHC-oligonucleotide complex 1105. Thehybridized oligonucleotides (Spacer C C C and Spacer rGrGrG) can then beextended in primer extension reactions such that constructs comprisingsequences that correspond to each of the two spatial barcode sequences(the spatial barcode associated with the capture probe, and the barcodeassociated with the MHC-oligonucleotide complex) are generated. In somecases, one or both of these corresponding sequences may be a complementof the original sequence in capture probe 1106 or capture agent barcodedomain 1101. In other embodiments, the capture probe and the captureagent barcode domain are ligated together. The resulting constructs canbe optionally further processed (e.g., to add any additional sequencesand/or for clean-up) and subjected to sequencing. As described elsewhereherein, a sequence derived from the capture probe 1106 spatial barcodesequence may be used to identify a feature and the sequence derived fromspatial barcode sequence on the capture agent barcode domain 1101 may beused to identify the particular peptide MHC complex 1104 bound on thesurface of the cell (e.g., when using MHC-peptide libraries forscreening immune cells or immune cell populations).

(C) Substrate

For the spatial array-based analytical methods described in thissection, the substrate functions as a support for direct or indirectattachment of capture probes to features of the array. In addition, insome embodiments, a substrate (e.g., the same substrate or a differentsubstrate) can be used to provide support to a biological sample,particularly, for example, a thin tissue section. Accordingly, a“substrate” is a support that is insoluble in aqueous liquid and whichallows for positioning of biological samples, analytes, features, and/orcapture probes on the substrate.

A wide variety of different substrates can be used for the foregoingpurposes. In general, a substrate can be any suitable support material.Exemplary substrates include, but are not limited to, glass, modifiedand/or functionalized glass, hydrogels, films, membranes, plastics(including e.g., acrylics, polystyrene, copolymers of styrene and othermaterials, polypropylene, polyethylene, polybutylene, polyurethanes,Teflon™, cyclic olefins, polyimides etc.), nylon, ceramics, resins,Zeonor, silica or silica-based materials including silicon and modifiedsilicon, carbon, metals, inorganic glasses, optical fiber bundles, andpolymers, such as polystyrene, cyclic olefin copolymers (COCs), cyclicolefin polymers (COPs), polypropylene, polyethylene and polycarbonate.

The substrate can also correspond to a flow cell. Flow cells can beformed of any of the foregoing materials, and can include channels thatpermit reagents, solvents, features, and molecules to pass through thecell.

Among the examples of substrate materials discussed above, polystyreneis a hydrophobic material suitable for binding negatively chargedmacromolecules because it normally contains few hydrophilic groups. Fornucleic acids immobilized on glass slides, by increasing thehydrophobicity of the glass surface the nucleic acid immobilization canbe increased. Such an enhancement can permit a relatively more denselypacked formation (e.g., provide improved specificity and resolution).

In some embodiments, a substrate is coated with a surface treatment suchas poly(L)-lysine. Additionally or alternatively, the substrate can betreated by silanation, e.g. with epoxysilane, amino-silane, and/or by atreatment with polyacrylamide.

The substrate can generally have any suitable form or format. Forexample, the substrate can be flat, curved, e.g. convexly or concavelycurved towards the area where the interaction between a biologicalsample, e.g. tissue sample, and the substrate takes place. In someembodiments, the substrate is a flat, e.g., planar, chip or slide. Thesubstrate can contain one or more patterned surfaces within thesubstrate (e.g., channels, wells, projections, ridges, divots, etc.).

A substrate can be of any desired shape. For example, a substrate can betypically a thin, flat shape (e.g., a square or a rectangle). In someembodiments, a substrate structure has rounded corners (e.g., forincreased safety or robustness). In some embodiments, a substratestructure has one or more cut-off corners (e.g., for use with a slideclamp or cross-table). In some embodiments, where a substrate structureis flat, the substrate structure can be any appropriate type of supporthaving a flat surface (e.g., a chip or a slide such as a microscopeslide).

Substrates can optionally include various structures such as, but notlimited to, projections, ridges, and channels. A substrate can bemicropatterned to limit lateral diffusion (e.g., to prevent overlap ofspatial barcodes). A substrate modified with such structures can bemodified to allow association of analytes, features (e.g., beads), orprobes at individual sites. For example, the sites where a substrate ismodified with various structures can be contiguous or non-contiguouswith other sites.

In some embodiments, the surface of a substrate can be modified so thatdiscrete sites are formed that can only have or accommodate a singlefeature. In some embodiments, the surface of a substrate can be modifiedso that features adhere to random sites.

In some embodiments, the surface of a substrate is modified to containone or more wells, using techniques such as (but not limited to)stamping techniques, microetching techniques, and molding techniques. Insome embodiments in which a substrate includes one or more wells, thesubstrate can be a concavity slide or cavity slide. For example, wellscan be formed by one or more shallow depressions on the surface of thesubstrate. In some embodiments, where a substrate includes one or morewells, the wells can be formed by attaching a cassette (e.g., a cassettecontaining one or more chambers) to a surface of the substratestructure.

In some embodiments, the structures of a substrate (e.g., wells) caneach bear a different capture probe. Different capture probes attachedto each structure can be identified according to the locations of thestructures in or on the surface of the substrate. Exemplary substratesinclude arrays in which separate structures are located on the substrateincluding, for example, those having wells that accommodate features.

In some embodiments, a substrate includes one or more markings on asurface of the substrate, e.g., to provide guidance for correlatingspatial information with the characterization of the analyte ofinterest. For example, a substrate can be marked with a grid of lines(e.g., to allow the size of objects seen under magnification to beeasily estimated and/or to provide reference areas for countingobjects). In some embodiments, fiducial markers can be included on thesubstrate. Such markings can be made using techniques including, but notlimited to, printing, sand-blasting, and depositing on the surface.

In some embodiments where the substrate is modified to contain one ormore structures, including but not limited to wells, projections,ridges, or markings, the structures can include physically alteredsites. For example, a substrate modified with various structures caninclude physical properties, including, but not limited to, physicalconfigurations, magnetic or compressive forces, chemicallyfunctionalized sites, chemically altered sites, and/or electrostaticallyaltered sites.

In some embodiments where the substrate is modified to contain variousstructures, including but not limited to wells, projections, ridges, ormarkings, the structures are applied in a pattern. Alternatively, thestructures can be randomly distributed.

In some embodiments, a substrate is treated in order to minimize orreduce non-specific analyte hybridization within or between features.For example, treatment can include coating the substrate with ahydrogel, film, and/or membrane that creates a physical barrier tonon-specific hybridization. Any suitable hydrogel can be used. Forexample, hydrogel matrices prepared according to the methods set forthin U.S. Pat. Nos. 6,391,937, 9,512,422, and 9,889,422, and U.S. Pat.Application Publication Nos. U.S. 2017/0253918 and U.S. 2018/0052081,can be used. The entire contents of each of the foregoing documents areincorporated herein by reference.

Treatment can include adding a functional group that is reactive orcapable of being activated such that it becomes reactive after receivinga stimulus (e.g., photoreactive). Treatment can include treating withpolymers having one or more physical properties (e.g., mechanical,electrical, magnetic, and/or thermal) that minimize non-specific binding(e.g., that activate a substrate at certain locations to allow analytehybridization at those locations).

The substrate (e.g., a bead or a feature on an array) can include tensto hundreds of thousands or millions of individual oligonucleotidemolecules (e.g., at least about 10,000, 50,000, 100,000, 500,000,1,000,000, 10,000,000, 100,000,000, 1,000,000,000, or 10,000,000,000oligonucleotide molecules).

In some embodiments, the surface of the substrate is coated with acell-permissive coating to allow adherence of live cells. A“cell-permissive coating” is a coating that allows or helps cells tomaintain cell viability (e.g., remain viable) on the substrate. Forexample, a cell-permissive coating can enhance cell attachment, cellgrowth, and/or cell differentiation, e.g., a cell-permissive coating canprovide nutrients to the live cells. A cell-permissive coating caninclude a biological material and/or a synthetic material. Non-limitingexamples of a cell-permissive coating include coatings that feature oneor more extracellular matrix (ECM) components (e.g., proteoglycans andfibrous proteins such as collagen, elastin, fibronectin and laminin),poly-lysine, poly(L)-ornithine, and/or a biocompatible silicone (e.g.,CYTOSOFT®). For example, a cell-permissive coating that includes one ormore extracellular matrix components can include collagen Type I,collagen Type II, collagen Type IV, elastin, fibronectin, laminin,and/or vitronectin. In some embodiments, the cell-permissive coatingincludes a solubilized basement membrane preparation extracted from theEngelbreth-Holm-Swarm (EHS) mouse sarcoma (e.g., MATRIGEL®). In someembodiments, the cell-permissive coating includes collagen. Acell-permissive coating can be used to culture adherent cells on aspatially-barcoded array, or to maintain cell viability of a tissuesample or section while in contact with a spatially-barcoded array.

Where the substrate includes a gel (e.g., a hydrogel or gel matrix),oligonucleotides within the gel can attach to the substrate. The terms“hydrogel” and “hydrogel matrix” are used interchangeably herein torefer to a macromolecular polymer gel including a network. Within thenetwork, some polymer chains can optionally be cross-linked, althoughcross-linking does not always occur.

In some embodiments, a hydrogel can include hydrogel subunits. A“hydrogel subunit” is a hydrophilic monomer, a molecular precursor, or apolymer that can be polymerized (e.g., cross-linked) to form athree-dimensional (3D) hydrogel network. The hydrogel subunits caninclude any convenient hydrogel subunits, such as, but not limited to,acrylamide, bis-acrylamide, polyacrylamide and derivatives thereof,poly(ethylene glycol) and derivatives thereof (e.g. PEG-acrylate(PEG-DA), PEG-RGD), gelatin-methacryloyl (GelMA), methacrylatedhyaluronic acid (MeHA), polyaliphatic polyurethanes, polyetherpolyurethanes, polyester polyurethanes, polyethylene copolymers,polyamides, polyvinyl alcohols, polypropylene glycol, polytetramethyleneoxide, polyvinyl pyrrolidone, polyacrylamide, poly(hydroxyethylacrylate), and poly(hydroxyethyl methacrylate), collagen, hyaluronicacid, chitosan, dextran, agarose, gelatin, alginate, protein polymers,methylcellulose, and the like, and combinations thereof.

In some embodiments, a hydrogel includes a hybrid material, e.g., thehydrogel material includes elements of both synthetic and naturalpolymers. Examples of suitable hydrogels are described, for example, inU.S. Pat. Nos. 6,391,937, 9,512,422, and 9,889,422, and in U.S. Pat.Application Publication Nos. 2017/0253918, 2018/0052081 and2010/0055733, the entire contents of each of which are incorporatedherein by reference.

In some embodiments, cross-linkers and/or initiators are added tohydrogel subunits. Examples of cross-linkers include, withoutlimitation, bis-acrylamide and diazirine. Examples of initiatorsinclude, without limitation, azobisisobutyronitrile (AIBN), riboflavin,and L-arginine. Inclusion of cross-linkers and/or initiators can lead toincreased covalent bonding between interacting biological macromoleculesin later polymerization steps.

In some embodiments, hydrogels can have a colloidal structure, such asagarose, or a polymer mesh structure, such as gelatin.

In some embodiments, some hydrogel subunits are polymerized (e.g.,undergo “formation”) covalently or physically cross-linked, to form ahydrogel network. For example, hydrogel subunits can be polymerized byany method including, but not limited to, thermal crosslinking, chemicalcrosslinking, physical crosslinking, ionic crosslinking,photo-crosslinking, irradiative crosslinking (e.g., x-ray, electronbeam), and combinations thereof. Techniques such as lithographicphotopolymerization can also be used to form hydrogels.

Polymerization methods for hydrogel subunits can be selected to formhydrogels with different properties (e.g., pore size, swellingproperties, biodegradability, conduction, transparency, and/orpermeability of the hydrogel). For example, a hydrogel can include poresof sufficient size to allow the passage of macromolecules, (e.g.,nucleic acids, proteins, chromatin, metabolites, gRNA, antibodies,carbohydrates, peptides, metabolites, and/or small molecules) into thesample (e.g., tissue section). It is known that pore size generallydecreases with increasing concentration of hydrogel subunits andgenerally increases with an increasing ratio of hydrogel subunits tocrosslinker. Therefore, a fixative/hydrogel composition can be preparedthat includes a concentration of hydrogel subunits that allows thepassage of such biological macromolecules.

In some embodiments, the hydrogel can form the substrate. In someembodiments, the substrate includes a hydrogel and one or more secondmaterials. In some embodiments, the hydrogel is placed on top of one ormore second materials. For example, the hydrogel can be preformed andthen placed on top of, underneath, or in any other configuration withone or more second materials. In some embodiments, hydrogel formationoccurs after contacting one or more second materials during formation ofthe substrate. Hydrogel formation can also occur within a structure(e.g., wells, ridges, projections, and/or markings) located on asubstrate.

In some embodiments, hydrogel formation on a substrate occurs before,contemporaneously with, or after features (e.g., beads) are attached tothe substrate. For example, when a capture probe is attached (e.g.,directly or indirectly) to a substrate, hydrogel formation can beperformed on the substrate already containing the capture probes.

In some embodiments, hydrogel formation occurs within a biologicalsample. In some embodiments, a biological sample (e.g., tissue section)is embedded in a hydrogel. In some embodiments, hydrogel subunits areinfused into the biological sample, and polymerization of the hydrogelis initiated by an external or internal stimulus.

In embodiments in which a hydrogel is formed within a biological sample,functionalization chemistry can be used. In some embodiments,functionalization chemistry includes hydrogel-tissue chemistry (HTC).Any hydrogel-tissue backbone (e.g., synthetic or native) suitable forHTC can be used for anchoring biological marcomolecules and modulatingfunctionalization. Non-limiting examples of methods using HTC backbonevariants include CLARITY, PACT, ExM, SWITCH and ePACT. In someembodiments, hydrogel formation within a biological sample is permanent.For example, biological macromolecules can permanently adhere to thehydrogel allowing multiple rounds of interrogation. In some embodiments,hydrogel formation within a biological sample is reversible.

In some embodiments, additional reagents are added to the hydrogelsubunits before, contemporaneously with, and/or after polymerization.For example, additional reagents can include but are not limited tooligonucleotides (e.g., capture probes), endonucleases to fragment DNA,fragmentation buffer for DNA, DNA polymerase enzymes, dNTPs used toamplify the nucleic acid and to attach the barcode to the amplifiedfragments. Other enzymes can be used, including without limitation, RNApolymerase, transposase, ligase, proteinase K, and DNAse. Additionalreagents can also include reverse transcriptase enzymes, includingenzymes with terminal transferase activity, primers, and switcholigonucleotides. In some embodiments, optical labels are added to thehydrogel subunits before, contemporaneously with, and/or afterpolymerization.

In some embodiments, HTC reagents are added to the hydrogel before,contemporaneously with, and/or after polymerization. In someembodiments, a cell labelling agent is added to the hydrogel before,contemporaneously with, and/or after polymerization. In someembodiments, a cell-penetrating agent is added to the hydrogel before,contemporaneously with, and/or after polymerization.

Hydrogels embedded within biological samples can be cleared using anysuitable method. For example, electrophoretic tissue clearing methodscan be used to remove biological macromolecules from thehydrogel-embedded sample. In some embodiments, a hydrogel-embeddedsample is stored before or after clearing of hydrogel, in a medium(e.g., a mounting medium, methylcellulose, or other semi-solid mediums).

A “conditionally removable coating” is a coating that can be removedfrom the surface of a substrate upon application of a releasing agent.In some embodiments, a conditionally removable coating includes ahydrogel as described herein, e.g., a hydrogel including apolypeptide-based material. Non-limiting examples of a hydrogelfeaturing a polypeptide-based material include a synthetic peptide-basedmaterial featuring a combination of spider silk and a trans-membranesegment of human muscle L-type calcium channel (e.g., PEPGEL®), anamphiphilic 16 residue peptide containing a repeatingarginine-alanine-aspartate-alanine sequence (RADARADARADARADA) (e.g.,PURAMATRIX®), EAK16 (AEAEAKAKAEAEAKAK), KLD12 (KLDLKLDLKLDL), andPGMATRIX™.

In some embodiments, the hydrogel in the conditionally removable coatingis a stimulus-responsive hydrogel. A stimulus-responsive hydrogel canundergo a gel-to-solution and/or gel-to-solid transition uponapplication of one or more external triggers (e.g., a releasing agent).See, e.g., Willner, Acc. Chem. Res. 50:657-658, 2017, which isincorporated herein by reference in its entirety. Non-limiting examplesof a stimulus-responsive hydrogel include a thermoresponsive hydrogel, apH-responsive hydrogel, a light-responsive hydrogel, a redox-responsivehydrogel, an analyte-responsive hydrogel, or a combination thereof. Insome embodiments, a stimulus-responsive hydrogel can be amulti-stimuli-responsive hydrogel.

A “releasing agent” or “external trigger” is an agent that allows forthe removal of a conditionally removable coating from a substrate whenthe releasing agent is applied to the conditionally removable coating.An external trigger or releasing agent can include physical triggerssuch as thermal, magnetic, ultrasonic, electrochemical, and/or lightstimuli as well as chemical triggers such as pH, redox reactions,supramolecular complexes, and/or biocatalytically driven reactions. Seee.g., Echeverria, et al., Gels (2018), 4, 54; doi:10.3390/gels4020054,which is incorporated herein by reference in its entirety. The type of“releasing agent” or “external trigger” can depend on the type ofconditionally removable coating. For example, a conditionally removablecoating featuring a redox-responsive hydrogel can be removed uponapplication of a releasing agent that includes a reducing agent such asdithiothreitol (DTT). As another example, a pH-responsive hydrogel canbe removed upon the application of a releasing agent that changes thepH.

(D) Arrays

In many of the methods described herein, features (as described furtherbelow) are collectively positioned on a substrate. An “array” is aspecific arrangement of a plurality of features that is either irregularor forms a regular pattern. Individual features in the array differ fromone another based on their relative spatial locations. In general, atleast two of the plurality of features in the array include a distinctcapture probe (e.g., any of the examples of capture probes describedherein).

Arrays can be used to measure large numbers of analytes simultaneously.In some embodiments, oligonucleotides are used, at least in part, tocreate an array. For example, one or more copies of a single species ofoligonucleotide (e.g., capture probe) can correspond to or be directlyor indirectly attached to a given feature in the array. In someembodiments, a given feature in the array includes two or more speciesof oligonucleotides (e.g., capture probes). In some embodiments, the twoor more species of oligonucleotides (e.g., capture probes) attacheddirectly or indirectly to a given feature on the array include a common(e.g., identical) spatial barcode.

A “feature” is an entity that acts as a support or repository forvarious molecular entities used in sample analysis. Examples of featuresinclude, but are not limited to, a bead, a spot of any two- orthree-dimensional geometry (e.g., an ink jet spot, a masked spot, asquare on a grid), a well, and a hydrogel pad. In some embodiments,features are directly or indirectly attached or fixed to a substrate. Insome embodiments, the features are not directly or indirectly attachedor fixed to a substrate, but instead, for example, are disposed withinan enclosed or partially enclosed three dimensional space (e.g., wellsor divots).

In addition to those above, a wide variety of other features can be usedto form the arrays described herein. For example, in some embodiments,features that are formed from polymers and/or biopolymers that are jetprinted, screen printed, or electrostatically deposited on a substratecan be used to form arrays. Jet printing of biopolymers is described,for example, in PCT Patent Application Publication No. WO 2014/085725.Jet printing of polymers is described, for example, in de Gans et al.,Adv Mater. 16(3): 203-213 (2004). Methods for electrostatic depositionof polymers and biopolymers are described, for example, in Hoyer et al.,Anal. Chem. 68(21): 3840-3844 (1996). The entire contents of each of theforegoing references are incorporated herein by reference.

As another example, in some embodiments, features are formed by metallicmicro- or nanoparticles. Suitable methods for depositing such particlesto form arrays are described, for example, in Lee et al., Beilstein J.Nanotechnol. 8: 1049-1055 (2017), the entire contents of which areincorporated herein by reference.

As a further example, in some embodiments, features are formed bymagnetic particles that are assembled on a substrate. Examples of suchparticles and methods for assembling arrays are described in Ye et al.,Scientific Reports 6: 23145 (2016), the entire contents of which areincorporated herein by reference.

As another example, in some embodiments, features correspond to regionsof a substrate in which one or more optical labels have beenincorporated, and/or which have been altered by a process such aspermanent photobleaching. Suitable substrates to implement features inthis manner include a wide variety of polymers, for example. Methods forforming such features are described, for example, in Moshrefzadeh etal., Appl. Phys. Lett. 62: 16 (1993), the entire contents of which areincorporated herein by reference.

As yet another example, in some embodiments, features can correspond tocolloidal particles assembled (e.g., via self-assembly) to form anarray. Suitable colloidal particles are described for example in Sharma,Resonance 23(3): 263-275 (2018), the entire contents of which areincorporated herein by reference.

As a further example, in some embodiments, features can be formed viaspot-array photopolymerization of a monomer solution on a substrate. Inparticular, two-photon and three-photon polymerization can be used tofabricate features of relatively small (e.g., sub-micron) dimensions.Suitable methods for preparing features on a substrate in this mannerare described for example in Nguyen et al., Materials Today 20(6):314-322 (2017), the entire contents of which are incorporated herein byreference.

In some embodiments, features are directly or indirectly attached orfixed to a substrate that is liquid permeable. In some embodiments,features are directly or indirectly attached or fixed to a substratethat is biocompatible. In some embodiments, features are directly orindirectly attached or fixed to a substrate that is a hydrogel.

FIG. 12 depicts an exemplary arrangement of barcoded features within anarray. From left to right, FIG. 12 shows (L) a slide including sixspatially-barcoded arrays, (C) an enlarged schematic of one of the sixspatially-barcoded arrays, showing a grid of barcoded features inrelation to a biological sample, and (R) an enlarged schematic of onesection of an array, showing the specific identification of multiplefeatures within the array (labelled as ID578, ID579, ID560, etc.).

As used herein, the term “bead array” refers to an array that includes aplurality of beads as the features in the array. In some embodiments,the beads are attached to a substrate. For example, the beads canoptionally attach to a substrate such as a microscope slide and inproximity to a biological sample (e.g., a tissue section that includescells). The beads can also be suspended in a solution and deposited on asurface (e.g., a membrane, a tissue section, or a substrate (e.g., amicroscope slide)).

Examples of arrays of beads on or within a substrate include beadslocated in wells such as the BeadChip array (available from IlluminaInc., San Diego, CA), arrays used in sequencing platforms from 454LifeSciences (a subsidiary of Roche, Basel, Switzerland), and array usedin sequencing platforms from Ion Torrent (a subsidiary of LifeTechnologies, Carlsbad, CA). Examples of bead arrays are described in,e.g., U.S. Pat. Nos. 6,266,459; 6,355,431; 6,770,441; 6,859,570;6,210,891; 6,258,568; and 6,274,320; U.S. Pat. Application PublicationNos. 2009/0026082; 2009/0127589; 2010/0137143; and 2010/0282617; and PCTPatent Application Publication Nos. WO 00/063437 and WO 2016/162309, theentire contents of each of which is incorporated herein by reference.

In some embodiments, the bead array includes a plurality of beads. Forexample, the bead array can include at least 10,000 beads (e.g., atleast 100,000 beads, at least 1,000,000 beads, at least 5,000,000 beads,at least 10,000,000 beads). In some embodiments, the plurality of beadsincludes a single type of beads (e.g., substantially uniform in size,shape, and other physical properties, such as translucence). In someembodiments, the plurality of beads includes two or more types ofdifferent beads.

In some embodiments, a bead array is formed when beads are embedded in ahydrogel layer where the hydrogel polymerizes and secures the relativebead positions. The bead-arrays can be pre-equilibrated and combinedwith reaction buffers and enzymes (e.g., reverse-transcription mix). Insome embodiments, the bead arrays are frozen.

A “flexible array” includes a plurality of spatially-barcoded featuresattached to, or embedded in, a flexible substrate (e.g., a membrane ortape) placed onto a biological sample. In some embodiments, a flexiblearray includes a plurality of spatially-barcoded features embeddedwithin a hydrogel matrix. To form such an array, features of amicroarray are copied into a hydrogel, and the size of the hydrogel isreduced by removing water. These steps can be performed multiple times.For example, in some embodiments, a method for preparing a high-densityspatially barcoded array can include copying a plurality of featuresfrom a microarray into a first hydrogel, where the first hydrogel is incontact with the microarray; reducing the size of the first hydrogelincluding the copied features by removing water, forming a firstshrunken hydrogel including the copied features; copying the features inthe first shrunken hydrogel into a second hydrogel, where the secondhydrogel is in contact with the first hydrogel; and reducing the size ofthe second hydrogel including the copied features by removing water,forming a second shrunken hydrogel including the copied features, thusgenerating a high-density spatially barcoded array. The result is ahigh-density flexible array including spatially-barcoded features.

In some embodiments, spatially-barcoded beads can be loaded onto asubstrate (e.g., a hydrogel) to produce a high-density self-assembledbead array.

Flexible arrays can be pre-equilibrated, combined with reaction buffersand enzymes at functional concentrations (e.g., a reverse-transcriptionmix). In some embodiments, the flexible bead-arrays can be stored forextended periods (e.g., days) or frozen until ready for use. In someembodiments, permeabilization of biological samples (e.g., a tissuesection) can be performed with the addition of enzymes/detergents priorto contact with the flexible array. The flexible array can be placeddirectly on the sample, or placed in indirect contact with thebiological sample (e.g., with an intervening layer or substance betweenthe biological sample and the flexible bead-array). In some embodiments,once a flexible array is applied to the sample, reverse transcriptionand targeted capture of analytes can be performed on solid microspheres,or circular beads of a first size and circular beads of a second size.

A “microcapillary array” is an arrayed series of features that arepartitioned by microcapillaries. A “microcapillary channel” is anindividual partition created by the microcapillaries. For example,microcapillary channels can be fluidically isolated from othermicrocapillary channels, such that fluid or other contents in onemicrocapillary channel in the array are separated from fluid or othercontents in a neighboring microcapillary channel in the array. Thedensity and order of the microcapillaries can be any suitable density ororder of discrete sites.

In some embodiments, microcapillary arrays are treated to generateconditions that facilitate loading. An example is the use of a coronawand (BD-20AC, Electro Technic Products) to generate a hydrophilicsurface. In some embodiments, a feature (e.g., a bead with capture probeattached) is loaded onto a microcapillary array such that the exactposition of the feature within the array is known. For example, acapture probe containing a spatial barcode can be placed into amicrocapillary channel so that the spatial barcode can enableidentification of the location from which the barcode sequence of thebarcoded nucleic acid molecule was derived.

In some embodiments, when random distribution is used to distributefeatures, empirical testing can be performed to generateloading/distribution conditions that facilitate a single feature permicrocapillary. In some embodiments, it can be desirable to achievedistribution conditions that facilitate only a single feature (e.g.,bead) per microcapillary channel. In some embodiments, it can bedesirable to achieve distribution conditions that facilitate more thanone feature (e.g., bead) per microcapillary channel, by flowing thefeatures through the microcapillary channel.

In some embodiments, the microcapillary array is placed in contact witha sample (e.g., on top or below) so that microcapillaries containing afeature (e.g., a bead, which can include a capture probe) are in contactwith the biological sample. In some embodiments, a biological sample isplaced onto an exposed side of a microcapillary array and mechanicalcompression is applied, moving the biological sample into themicrocapillary channel to create a fluidically isolated reaction chambercontaining the biological sample.

In some embodiments, a biological sample is partitioned by contacting amicrocapillary array to the biological sample, thereby creatingmicrocapillary channels including a bead and a portion of the biologicalsample. In some embodiments, a portion of a biological sample containedin a microcapillary channel is one or more cells. In some embodiments, afeature is introduced into a microcapillary array by flow after one ormore cells are added to a microcapillary channel.

In some embodiments, reagents are added to the microcapillary array. Theadded reagents can include enzymatic reagents, and reagent mixtures forperforming amplification of a nucleic acid. In some embodiments, thereagents include a reverse transcriptase, a ligase, one or morenucleotides, and any combinations thereof. One or more microcapillarychannels can be sealed after reagents are added to the microcapillarychannels, e.g. using silicone oil, mineral oil, a nonporous material, orlid.

In some embodiments, a reagent solution is removed from eachmicrocapillary channel following an incubation for an amount of time andat a certain temperature or range of temperatures, e.g., following ahybridization or an amplification reaction. Reagent solutions can beprocessed individually for sequencing, or pooled for sequencinganalysis.

In some embodiments, some or all features in an array include a captureprobe. In some embodiments, an array can include a capture probeattached directly or indirectly to the substrate.

The capture probe includes a capture domain (e.g., a nucleotidesequence) that can specifically bind (e.g., hybridize) to a targetanalyte (e.g., mRNA, DNA, or protein) within a sample. In someembodiments, the binding of the capture probe to the target (e.g.,hybridization) can be detected and quantified by detection of a visualsignal, e.g. a fluorophore, a heavy metal (e.g., silver ion), orchemiluminescent label, which has been incorporated into the target. Insome embodiments, the intensity of the visual signal correlates with therelative abundance of each analyte in the biological sample. Since anarray can contain thousands or millions of capture probes (or more), anarray of features with capture probes can interrogate many analytes inparallel.

In some embodiments, a substrate includes one or more capture probesthat are designed to capture analytes from one or more organisms. In anon-limiting example, a substrate can contain one or more capture probesdesigned to capture mRNA from one organism (e.g., a human) and one ormore capture probes designed to capture DNA from a second organism(e.g., a bacterium).

The capture probes can be attached to a substrate or feature using avariety of techniques. In some embodiments, the capture probe isdirectly attached to a feature that is fixed on an array. In someembodiments, the capture probes are immobilized to a substrate bychemical immobilization. For example, a chemical immobilization can takeplace between functional groups on the substrate and correspondingfunctional elements on the capture probes. Exemplary correspondingfunctional elements in the capture probes can either be an inherentchemical group of the capture probe, e.g. a hydroxyl group, or afunctional element can be introduced on to the capture probe. An exampleof a functional group on the substrate is an amine group. In someembodiments, the capture probe to be immobilized includes a functionalamine group or is chemically modified in order to include a functionalamine group. Means and methods for such a chemical modification are wellknown in the art.

In some embodiments, the capture probe is a nucleic acid. In someembodiments, the capture probe is immobilized on the feature or thesubstrate via its 5′ end. In some embodiments, the capture probe isimmobilized on a feature or a substrate via its 5′ end and includes fromthe 5′ to 3′ end: one or more barcodes (e.g., a spatial barcode and/or aUMI) and one or more capture domains. In some embodiments, the captureprobe is immobilized on a feature via its 5′ end and includes from the5′ to 3′ end: one barcode (e.g., a spatial barcode or a UMI) and onecapture domain. In some embodiments, the capture probe is immobilized ona feature or a substrate via its 5′ end and includes from the 5′ to 3′end: a cleavage domain, a functional domain, one or more barcodes (e.g.,a spatial barcode and/or a UMI), and a capture domain.

In some embodiments, the capture probe is immobilized on a feature or asubstrate via its 5′ end and includes from the 5′ to 3′ end: a cleavagedomain, a functional domain, one or more barcodes (e.g., a spatialbarcode and/or a UMI), a second functional domain, and a capture domain.In some embodiments, the capture probe is immobilized on a feature or asubstrate via its 5′ end and includes from the 5′ to 3′ end: a cleavagedomain, a functional domain, a spatial barcode, a UMI, and a capturedomain. In some embodiments, the capture probe is immobilized on afeature or a substrate via its 5′ end and does not include a spatialbarcode. In some embodiments, the capture probe is immobilized on afeature or a substrate via its 5′ end and does not include a UMI. Insome embodiments, the capture probe includes a sequence for initiating asequencing reaction.

In some embodiments, the capture probe is immobilized on a feature or asubstrate via its 3′ end. In some embodiments, the capture probe isimmobilized on a feature or a substrate via its 3′ end and includes fromthe 3′ to 5′ end: one or more barcodes (e.g., a spatial barcode and/or aUMI) and one or more capture domains. In some embodiments, the captureprobe is immobilized on a feature or a substrate via its 3′ end andincludes from the 3′ to 5′ end: one barcode (e.g., a spatial barcode ora UMI) and one capture domain. In some embodiments, the capture probe isimmobilized on a feature or a substrate via its 3′ end and includes fromthe 3′ to 5′ end: a cleavage domain, a functional domain, one or morebarcodes (e.g., a spatial barcode and/or a UMI), and a capture domain.In some embodiments, the capture probe is immobilized on a feature or asubstrate via its 3′ end and includes from the 3′ to 5′ end: a cleavagedomain, a functional domain, a spatial barcode, a UMI, and a capturedomain.

The localization of the functional group within the capture probe to beimmobilized can be used to control and shape the binding behavior and/ororientation of the capture probe, e.g. the functional group can beplaced at the 5′ or 3′ end of the capture probe or within the sequenceof the capture probe. In some embodiments, a capture probe can furtherinclude a substrate (e.g., a support attached to the capture probe, asupport attached to the feature, or a support attached to thesubstrate). A typical substrate for a capture probe to be immobilizedincludes moieties which are capable of binding to such capture probes,e.g., to amine-functionalized nucleic acids. Examples of such substratesare carboxy, aldehyde, or epoxy supports.

In some embodiments, the substrates on which capture probes can beimmobilized can be chemically activated, e.g. by the activation offunctional groups, available on the substrate. The term “activatedsubstrate” relates to a material in which interacting or reactivechemical functional groups are established or enabled by chemicalmodification procedures. For example, a substrate including carboxylgroups can be activated before use. Furthermore, certain substratescontain functional groups that can react with specific moieties alreadypresent in the capture probes.

In some embodiments, a covalent linkage is used to directly couple acapture probe to a substrate. In some embodiments a capture probe isindirectly coupled to a substrate through a linker separating the“first” nucleotide of the capture probe from the substrate, i.e., achemical linker. In some embodiments, a capture probe does not binddirectly to the array, but interacts indirectly, for example by bindingto a molecule which itself binds directly or indirectly to the array. Insome embodiments, the capture probe is indirectly attached to asubstrate (e.g., via a solution including a polymer).

In some embodiments where the capture probe is immobilized on thefeature of the array indirectly, e.g. via hybridization to a surfaceprobe capable of binding the capture probe, the capture probe canfurther include an upstream sequence (5′ to the sequence that hybridizesto the nucleic acid, e.g. RNA of the tissue sample) that is capable ofhybridizing to 5′ end of the surface probe. Alone, the capture domain ofthe capture probe can be seen as a capture domain oligonucleotide, whichcan be used in the synthesis of the capture probe in embodiments wherethe capture probe is immobilized on the array indirectly.

In some embodiments, a substrate is comprised of an inert material ormatrix (e.g., glass slides) that has been functionalization by, forexample, treatment with a material comprising reactive groups whichenable immobilization of capture probes. See, for example, WO2017/019456, the entire contents of which are herein incorporated byreference. Non-limiting examples include polyacrylamide hydrogelssupported on an inert substrate (e.g., glass slide; see WO 2005/065814and U.S. Pat. Application No. 2008/0280773, the entire contents of whichare incorporated herein by reference).

In some embodiments, functionalized biomolecules (e.g., capture probes)are immobilized on a functionalized substrate using covalent methods.Methods for covalent attachment include, for example, condensation ofamines and activated carboxylic esters (e.g., N-hydroxysuccinimideesters); condensation of amine and aldehydes under reductive aminationconditions; and cycloaddition reactions such as the Diels-Alder [4+2]reaction, 1,3-dipolar cycloaddition reactions, and [2+2] cycloadditionreactions. Methods for covalent attachment also include, for example,click chemistry reactions, including [3+2] cycloaddition reactions(e.g., Huisgen 1,3-dipolar cycloaddition reaction andcopper(I)-catalyzed azide-alkyne cycloaddition (CuAAC)); thiol-enereactions; the Diels-Alder reaction and inverse electron demandDiels-Alder reaction; [4+1] cycloaddition of isonitriles and tetrazines;and nucleophilic ring-opening of small carbocycles (e.g., epoxideopening with amino oligonucleotides). Methods for covalent attachmentalso include, for example, maleimides and thiols; and para-nitrophenylester-functionalized oligonucleotides and polylysine-functionalizedsubstrate. Methods for covalent attachment also include, for example,disulfide reactions; radical reactions (see, e.g., U.S. Pat. No.5,919,626, the entire contents of which are herein incorporated byreference); and hydrazide-functionalized substrate (e.g., wherein thehydrazide functional group is directly or indirectly attached to thesubstrate) and aldehyde-functionalized oligonucleotides (see, e.g.,Yershov et al. (1996) Proc. Natl. Acad. Sci. USA 93, 4913-4918, theentire contents of which are herein incorporated by reference).

In some embodiments, functionalized biomolecules (e.g., capture probes)are immobilized on a functionalized substrate using photochemicalcovalent methods. Methods for photochemical covalent attachment include,for example, immobilization of antraquinone-conjugated oligonucleotides(see, e.g., Koch et al. (2000) Bioconjugate Chem. 11, 474-483, theentire contents of which are herein incorporated by reference).

In some embodiments, functionalized biomolecules (e.g., capture probesare immobilized on a functionalized substrate using non-covalentmethods. Methods for non-covalent attachment include, for example,biotin-functionalized oligonucleotides and streptavidin-treatedsubstrates (see, e.g., Holmstrøm et al. (1993) Analytical Biochemistry209, 278-283 and Gilles et al. (1999) Nature Biotechnology 17, 365-370,the entire contents of which are herein incorporated by reference).

In some embodiments, an oligonucleotide (e.g., a capture probe) can beattached to a substrate or feature according to the methods set forth inU.S. Pat. Nos. 6,737,236, 7,259,258, 7,375,234, 7,427,678, 5,610,287,5,807,522, 5,837,860, and 5,472,881; U.S. Pat. Application PublicationNos. 2008/0280773 and 2011/0059865; Shalon et al. (1996) GenomeResearch, 639-645; Rogers et al. (1999) Analytical Biochemistry 266,23-30; Stimpson et al. (1995) Proc. Natl. Acad. Sci. USA 92, 6379-6383;Beattie et al. (1995) Clin. Chem. 45, 700-706; Lamture et al. (1994)Nucleic Acids Research 22, 2121-2125; Beier et al. (1999) Nucleic AcidsResearch 27, 1970-1977; Joos et al. (1997) Analytical Biochemistry 247,96-101; Nikiforov et al. (1995) Analytical Biochemistry 227, 201-209;Timofeev et al. (1996) Nucleic Acids Research 24, 3142-3148; Chrisey etal. (1996) Nucleic Acids Research 24, 3031-3039; Guo et al. (1994)Nucleic Acids Research 22, 5456-5465; Running and Urdea (1990)BioTechniques 8, 276-279; Fahy et al. (1993) Nucleic Acids Research 21,1819-1826; Zhang et al. (1991) 19, 3929-3933; and Rogers et al. (1997)Gene Therapy 4, 1387-1392. The entire contents of each of the foregoingdocuments are incorporated herein by reference.

Arrays can be prepared by a variety of methods. In some embodiments,arrays are prepared through the synthesis (e.g., in-situ synthesis) ofoligonucleotides on the array, or by jet printing or lithography. Forexample, light-directed synthesis of high-density DNA oligonucleotidescan be achieved by photolithography or solid-phase DNA synthesis. Toimplement photolithographic synthesis, synthetic linkers modified withphotochemical protecting groups can be attached to a substrate and thephotochemical protecting groups can be modified using aphotolithographic mask (applied to specific areas of the substrate) andlight, thereby producing an array having localized photo-deprotection.Many of these methods are known in the art, and are described e.g., inMiller et al., “Basic concepts of microarrays and potential applicationsin clinical microbiology.” Clinical microbiology reviews 22.4 (2009):611-633; US201314111482A; US9593365B2; US2019203275; and WO2018091676,which are incorporated herein by reference in the entirety.

In some embodiments, the arrays are “spotted” or “printed” witholigonucleotides and these oligonucleotides (e.g., capture probes) arethen attached to the substrate. The oligonucleotides can be applied byeither noncontact or contact printing. A noncontact printer can use thesame method as computer printers (e.g., bubble jet or inkjet) to expelsmall droplets of probe solution onto the substrate. The specializedinkjet-like printer can expel nanoliter to picoliter volume droplets ofoligonucleotide solution, instead of ink, onto the substrate. In contactprinting, each print pin directly applies the oligonucleotide solutiononto a specific location on the surface. The oligonucleotides can beattached to the substrate surface by the electrostatic interaction ofthe negative charge of the phosphate backbone of the DNA with apositively charged coating of the substrate surface or byUV-cross-linked covalent bonds between the thymidine bases in the DNAand amine groups on the treated substrate surface. In some embodiments,the substrate is a glass slide. In some embodiments, theoligonucleotides (e.g., capture probes) are attached to the substrate bya covalent bond to a chemical matrix, e.g. epoxy-silane, amino-silane,lysine, polyacrylamide, etc.

The arrays can also be prepared by in situ-synthesis. In someembodiments, these arrays can be prepared using photolithography. Themethod typically relies on UV masking and light-directed combinatorialchemical synthesis on a substrate to selectively synthesize probesdirectly on the surface of the array, one nucleotide at a time per spot,for many spots simultaneously. In some embodiments, a substrate containscovalent linker molecules that have a protecting group on the free endthat can be removed by light. UV light is directed through aphotolithographic mask to deprotect and activate selected sites withhydroxyl groups that initiate coupling with incoming protectednucleotides that attach to the activated sites. The mask is designed insuch a way that the exposure sites can be selected, and thus specify thecoordinates on the array where each nucleotide can be attached. Theprocess can be repeated, a new mask is applied activating different setsof sites and coupling different bases, allowing arbitraryoligonucleotides to be constructed at each site. This process can beused to synthesize hundreds of thousands of different oligonucleotides.In some embodiments, maskless array synthesizer technology can be used.It uses an array of programmable micromirrors to create digital masksthat reflect the desired pattern of UV light to deprotect the features.

In some embodiments, the inkjet spotting process can also be used forin-situ oligonucleotide synthesis. The different nucleotide precursorsplus catalyst can be printed on the substrate, and are then combinedwith coupling and deprotection steps. This method relies on printingpicoliter volumes of nucleotides on the array surface in repeated roundsof base-by-base printing that extends the length of the oligonucleotideprobes on the array.

Arrays can also be prepared by active hybridization via electric fieldsto control nucleic acid transport. Negatively charged nucleic acids canbe transported to specific sites, or features, when a positive currentis applied to one or more test sites on the array. The surface of thearray can contain a binding molecule, e.g., streptavidin, which allowsfor the formation of bonds (e.g., streptavidin-biotin bonds) onceelectronically addressed biotinylated probes reach their targetedlocation. The positive current is then removed from the active features,and new test sites can be activated by the targeted application of apositive current. The process are repeated until all sites on the arrayare covered.

An array for spatial analysis can be generated by various methods asdescribed herein. In some embodiments, the array has a plurality ofcapture probes comprising spatial barcodes. These spatial barcodes andtheir relationship to the locations on the array can be determined. Insome cases, such information is readily available, because theoligonucleotides are spotted, printed, or synthesized on the array witha pre-determined pattern. In some cases, the spatial barcode can bedecoded by methods described herein, e.g., by in-situ sequencing, byvarious labels associated with the spatial barcodes etc. In someembodiments, an array can be used as a template to generate a daughterarray. Thus, the spatial barcode can be transferred to the daughterarray with a known pattern.

In some embodiments, an array comprising barcoded probes can begenerated through ligation of a plurality of oligonucleotides. In someinstances, an oligonucleotide of the plurality contains a portion of abarcode, and the complete barcode is generated upon ligation of theplurality of oligonucleotides. For example, a first oligonucleotidecontaining a first portion of a barcode can be attached to a substrate(e.g., using any of the methods of attaching an oligonucleotide to asubstrate described herein), and a second oligonucleotide containing asecond portion of the barcode can then be ligated onto the firstoligonucleotide to generate a complete barcode. Different combinationsof the first, second and any additional portions of a barcode can beused to increase the diversity of the barcodes. In instances where thesecond oligonucleotide is also attached to the substrate prior toligation, the first and/or the second oligonucleotide can be attached tothe substrate via a surface linker which contains a cleavage site. Uponligation, the ligated oligonucleotide is linearized by cleaving at thecleavage site.

To increase the diversity of the barcodes, a plurality of secondoligonucleotides comprising two or more different barcode sequences canbe ligated onto a plurality of first oligonucleotides that comprise thesame barcode sequence, thereby generating two or more different speciesof barcodes. To achieve selective ligation, a first oligonucleotideattached to a substrate containing a first portion of a barcode caninitially be protected with a protective group (e.g., a photocleavableprotective group), and the protective group can be removed prior toligation between the first and second oligonucleotide. In instanceswhere the barcoded probes on an array are generated through ligation oftwo or more oligonucleotides, a concentration gradient of theoligonucleotides can be applied to a substrate such that differentcombinations of the oligonucleotides are incorporated into a barcodedprobe depending on its location on the substrate.

Barcoded probes on an array can also be generated by adding singlenucleotides to existing oligonucleotides on an array, for example, usingpolymerases that function in a template-independent manner. Singlenucleotides can be added to existing oligonucleotides in a concentrationgradient, thereby generating probes with varying length, depending onthe location of the probes on the array.

Arrays can also be prepared by modifying existing arrays, for example,by modifying the oligonucleotides attached to the arrays. For instance,probes can be generated on an array that comprises oligonucleotides thatare attached to the array at the 3′ end and have a free 5′ end. Theoligonucleotides can be in situ synthesized oligonucleotides, and caninclude a barcode. The length of the oligonucleotides can be less than50 nucleotides (nts) (e.g., less than 45, 40, 35, 30, 25, 20, 15, or 10nts). To generate probes using these oligonucleotides, a primercomplementary to a portion of an oligonucleotide (e.g., a constantsequence shared by the oligonucleotides) can be used to hybridize withthe oligonucleotide and extend (using the oligonucleotide as a template)to form a duplex and to create a 3′ overhang. The 3′ overhang thusallows additional nucleotides or oligonucleotides to be added on to theduplex. A capture probe can be generated by, for instance, adding one ormore oligonucleotides to the end of the 3′ overhang (e.g., via splintoligonucleotide mediated ligation), where the added oligonucleotides caninclude the sequence or a portion of the sequence of a capture domain.

In instances where the oligonucleotides on an existing array include arecognition sequence that can hybridize with a splint oligonucleotide,probes can also be generated by directly ligating additionaloligonucleotides onto the existing oligonucleotides via the splintoligonucleotide. The recognition sequence can at the free 5′ end or thefree 3′ end of an oligonucleotide on the existing array. Recognitionsequences useful for the methods of the present disclosure may notcontain restriction enzyme recognition sites or secondary structures(e.g., hairpins), and may include high contents of Guanine and Cytosinenucleotides and thus have high stability.

Bead arrays can be generated by attaching beads (e.g., barcoded beads)to a substrate in a regular pattern, or an irregular arrangement. Beadscan be attached to selective regions on a substrate by, e.g.,selectively activating regions on the substrate to allow for attachmentof the beads. Activating selective regions on the substrate can includeactivating a coating (e.g., a photocleavable coating) or a polymer thatis applied on the substrate. Beads can be attached iteratively, e.g., asubset of the beads can be attached at one time, and the same processcan be repeated to attach the remaining beads. Alternatively, beads canbe attached to the substrate all in one step.

Barcoded beads, or beads comprising a plurality of barcoded probes, canbe generated by first preparing a plurality of barcoded probes on asubstrate, depositing a plurality of beads on the substrate, andgenerating probes attached to the beads using the probes on thesubstrate as a template.

Large scale commercial manufacturing methods allow for millions ofoligonucleotides to be attached to an array. Commercially availablearrays include those from Roche NimbleGen, Inc., (Wisconsin) andAffymetrix (ThermoFisher Scientific).

In some embodiments, arrays can be prepared according to the methods setforth in WO 2012/140224, WO 2014/060483, WO 2016/162309, WO 2017/019456,WO 2018/091676, and WO 2012/140224, and U.S. Pat. Application No.2018/0245142. The entire contents of the foregoing documents are hereinincorporated by reference.

In some embodiments, a feature on the array includes a bead. In someembodiments, two or more beads are dispersed onto a substrate to createan array, where each bead is a feature on the array. Beads canoptionally be dispersed into wells on a substrate, e.g., such that onlya single bead is accommodated per well.

A “bead” is a particle. A bead can be porous, non-porous, solid,semi-solid, and/or a combination thereof. In some embodiments, a beadcan be dissolvable, disruptable, and/or degradable, whereas in certainembodiments, a bead is not degradable.

A bead can generally be of any suitable shape. Examples of bead shapesinclude, but are not limited to, spherical, non-spherical, oval, oblong,amorphous, circular, cylindrical, and variations thereof. A crosssection (e.g., a first cross-section) can correspond to a diameter ormaximum cross-sectional dimension of the bead. In some embodiments, thebead can be approximately spherical. In such embodiments, the firstcross-section can correspond to the diameter of the bead. In someembodiments, the bead can be approximately cylindrical. In suchembodiments, the first cross-section can correspond to a diameter,length, or width along the approximately cylindrical bead.

Beads can be of uniform size or heterogeneous size. “Polydispersity”generally refers to heterogeneity of sizes of molecules or particles.The polydispersity index (PDI) of a bead can be calculated using theequation PDI = Mw/Mn, where Mw is the weight-average molar mass and Mnis the number-average molar mass. In certain embodiments, beads can beprovided as a population or plurality of beads having a relativelymonodisperse size distribution. Where it can be desirable to providerelatively consistent amounts of reagents, maintaining relativelyconsistent bead characteristics, such as size, can contribute to theoverall consistency.

In some embodiments, the beads provided herein can have sizedistributions that have a coefficient of variation in theircross-sectional dimensions of less than 50%, less than 40%, less than30%, less than 20%, less than 15%, less than 10%, less than 5%, orlower. In some embodiments, a plurality of beads provided herein has apolydispersity index of less than 50%, less than 45%, less than 40%,less than 35%, less than 30%, less than 25%, less than 20%, less than15%, less than 10%, less than 5%, or lower.

In some embodiments, the bead can have a diameter or maximum dimensionno larger than 100 µm (e.g., no larger than 95 µm, 90 µm, 85 µm, 80 µm,75 µm, 70 µm, 65 µm, 60 µm, 55 µm, 50 µm, 45 µm, 40 µm, 35 µm, 30 µm, 25µm, 20 µm, 15 µm, 14 µm, 13 µm, 12 µm, 11 µm, 10 µm, 9 µm, 8 µm, 7 µm, 6µm, 5 µm, 4 µm, 3 µm, 2 µm, or 1 µm.)

In some embodiments, a plurality of beads has an average diameter nolarger than 100 µm. In some embodiments, a plurality of beads has anaverage diameter or maximum dimension no larger than 95 µm, 90 µm, 85µm, 80 µm, 75 µm, 70 µm, 65 µm, 60 µm, 55 µm, 50 µm, 45 µm, 40 µm, 35µm, 30 µm, 25 µm, 20 µm, 15 µm, 14 µm, 13 µm, 12 µm, 11 µm, 10 µm, 9 µm,8 µm, 7 µm, 6 µm, 5 µm, 4 µm, 3 µm, 2 µm, or 1 µm.

In some embodiments, the volume of the bead can be at least about 1 µm³,e.g., at least 1 µm³, 2 µm³, 3 µm³, 4 µm³, 5 µm³, 6 µm³, 7 µm³, 8 µm³, 9µm³, 10 µm³, 12 µm³, 14 µm³, 16 µm³, 18 µm³, 20 µm³, 25 µm³, 30 µm³, 35µm³, 40 µm³, 45 µm³, 50 µm³, 55 µm³, 60 µm³, 65 µm³, 70 µm³, 75 µm³, 80µm³, 85 µm³, 90 µm³, 95 µm³, 100 µm³, 125 µm³, 150 µm³, 175 µm³, 200µm³, 250 µm³, 300 µm³, 350 µm³, 400 µm³, 450 µm³, µm³, 500 µm³, 550 µm³,600 µm³, 650 µm³, 700 µm³, 750 µm³, 800 µm³, 850 µm³, 900 µm³, 950 µm³,1000 µm³, 1200 µm³, 1400 µm³, 1600 µm³, 1800 µm³, 2000 µm³, 2200 µm³,2400 µm³, 2600 µm³, 2800 µm³, 3000 µm³, or greater.

In some embodiments, the bead can have a volume of between about 1 µm³and 100 µm³, such as between about 1 µm³ and 10 µm³, between about 10µm³ and 50 µm³, or between about 50 µm³ and 100 µm³. In someembodiments, the bead can include a volume of between about 100 µm³ and1000 µm³, such as between about 100 µm³ and 500 µm³ or between about 500µm³ and 1000 µm³. In some embodiments, the bead can include a volumebetween about 1000 µm³ and 3000 µm³, such as between about 1000 µm³ and2000 µm³ or between about 2000 µm³ and 3000 µm³. In some embodiments,the bead can include a volume between about 1 µm³ and 3000 µm³, such asbetween about 1 µm³ and 2000 µm³, between about 1 µm³ and 1000 µm³,between about 1 µm³ and 500 µm³, or between about 1 µm³ and 250 µm³.

The bead can include one or more cross-sections that can be the same ordifferent. In some embodiments, the bead can have a first cross-sectionthat is different from a second cross-section. The bead can have a firstcross-section that is at least about 0.0001 micrometer, 0.001micrometer, 0.01 micrometer, 0.1 micrometer, or 1 micrometer. In someembodiments, the bead can include a cross-section (e.g., a firstcross-section) of at least about 1 micrometer (µm), 2 µm, 3 µm, 4 µm, 5µm, 6 µm, 7 µm, 8 µm, 9 µm, 10 µm, 11 µm, 12 µm, 13 µm, 14 µm, 15 µm, 16µm, 17 µm, 18 µm, 19 µm, 20 µm, 25 µm, 30 µm, 35 µm, 40 µm, 45 µm, 50µm, 55 µm, 60 µm, 65 µm, 70 µm, 75 µm, 80 µm, 85 µm, 90 µm, 100 µm, 120µm, 140 µm, 160 µm, 180 µm, 200 µm, 250 µm, 300 µm, 350 µm, 400 µm, 450µm, 500 µm, 550 µm, 600 µm, 650 µm, 700 µm, 750 µm, 800 µm, 850 µm, 900µm, 950 µm, 1 millimeter (mm), or greater. In some embodiments, the beadcan include a cross-section (e.g., a first cross-section) of betweenabout 1 µm and 500 µm, such as between about 1 µm and 100 µm, betweenabout 100 µm and 200 µm, between about 200 µm and 300 µm, between about300 µm and 400 µm, or between about 400 µm and 500 µm. For example, thebead can include a cross-section (e.g., a first cross-section) ofbetween about 1 µm and 100 µm. In some embodiments, the bead can have asecond cross-section that is at least about 1 µm. For example, the beadcan include a second cross-section of at least about 1 micrometer (µm),2 µm, 3 µm, 4 µm, 5 µm, 6 µm, 7 µm, 8 µm, 9 µm, 10 µm, 11 µm, 12 µm, 13µm, 14 µm, 15 µm, 16 µm, 17 µm, 18 µm, 19 µm, 20 µm, 25 µm, 30 µm, 35µm, 40 µm, 45 µm, 50 µm, 55 µm, 60 µm, 65 µm, 70 µm, 75 µm, 80 µm, 85µm, 90 µm, 100 µm, 120 µm, 140 µm, 160 µm, 180 µm, 200 µm, 250 µm, 300µm, 350 µm, 400 µm, 450 µm, 500 µm, 550 µm, 600 µm, 650 µm, 700 µm, 750µm, 800 µm, 850 µm, 900 µm, 950 µm, 1 millimeter (mm), or greater. Insome embodiments, the bead can include a second cross-section of betweenabout 1 µm and 500 µm, such as between about 1 µm and 100 µm, betweenabout 100 µm and 200 µm, between about 200 µm and 300 µm, between about300 µm and 400 µm, or between about 400 µm and 500 µm. For example, thebead can include a second cross-section of between about 1 µm and 100µm.

In some embodiments, beads can be of a nanometer scale (e.g., beads canhave a diameter or maximum cross-sectional dimension of about 100nanometers (nm) to about 900 nanometers (nm) (e.g., 850 nm or less, 800nm or less, 750 nm or less, 700 nm or less, 650 nm or less, 600 nm orless, 550 nm or less, 500 nm or less, 450 nm or less, 400 nm or less,350 nm or less, 300 nm or less, 250 nm or less, 200 nm or less, 150 nmor less). A plurality of beads can have an average diameter or averagemaximum cross-sectional dimension of about 100 nanometers (nm) to about900 nanometers (nm) (e.g., 850 nm or less, 800 nm or less, 750 nm orless, 700 nm or less, 650 nm or less, 600 nm or less, 550 nm or less,500 nm or less, 450 nm or less, 400 nm or less, 350 nm or less, 300 nmor less, 250 nm or less, 200 nm or less, 150 nm or less). In someembodiments, a bead has a diameter or size that is about the size of asingle cell (e.g., a single cell under evaluation).

In some embodiments, the bead can be a gel bead. A “gel” is a semi-rigidmaterial permeable to liquids and gases. Exemplary gels include, but arenot limited to, those having a colloidal structure, such as agarose;polymer mesh structures, such as gelatin; hydrogels; and cross-linkedpolymer structures, such as polyacrylamide, SFA (see, for example, U.S.Pat. Application Publication No. 2011/0059865, which is incorporatedherein by reference in its entirety) and PAZAM (see, for example, U.S.Pat. Application Publication No. 2014/0079923, which is incorporatedherein by reference in its entirety).

A gel can be formulated into various shapes and dimensions depending onthe context of intended use. In some embodiments, a gel is prepared andformulated as a gel bead (e.g., a gel bead including capture probesattached or associated with the gel bead). A gel bead can be a hydrogelbead. A hydrogel bead can be formed from molecular precursors, such as apolymeric or monomeric species.

In some embodiments, a hydrogel bead can include a polymer matrix (e.g.,a matrix formed by polymerization or cross-linking). A polymer matrixcan include one or more polymers (e.g., polymers having differentfunctional groups or repeat units). Cross-linking can be via covalent,ionic, and/or inductive interactions, and/or physical entanglement.

A semi-solid bead can be a liposomal bead.

Solid beads can include metals including, without limitation, ironoxide, gold, and silver. In some embodiments, the bead can be a silicabead. In some embodiments, the bead can be rigid. In some embodiments,the bead can be flexible and/or compressible.

The bead can be a macromolecule. The bead can be formed of nucleic acidmolecules bound together. The bead can be formed via covalent ornon-covalent assembly of molecules (e.g., macromolecules), such asmonomers or polymers. Polymers or monomers can be natural or synthetic.Polymers or monomers can be or include, for example, nucleic acidmolecules (e.g., DNA or RNA).

A bead can be rigid, or flexible and/or compressible. A bead can includea coating including one or more polymers. Such a coating can bedisruptable or dissolvable. In some embodiments, a bead includes aspectral or optical label (e.g., dye) attached directly or indirectly(e.g., through a linker) to the bead. For example, a bead can beprepared as a colored preparation (e.g., a bead exhibiting a distinctcolor within the visible spectrum) that can change color (e.g.,colorimetric beads) upon application of a desired stimulus (e.g., heatand/or chemical reaction) to form differently colored beads (e.g.,opaque and/or clear beads).

A bead can include natural and/or synthetic materials. For example, abead can include a natural polymer, a synthetic polymer or both naturaland synthetic polymers. Examples of natural polymers include, withoutlimitation, proteins, sugars such as deoxyribonucleic acid, rubber,cellulose, starch (e.g., amylose, amylopectin), enzymes,polysaccharides, silks, polyhydroxyalkanoates, chitosan, dextran,collagen, carrageenan, ispaghula, acacia, agar, gelatin, shellac,sterculia gum, xanthan gum, corn sugar gum, guar gum, gum karaya,agarose, alginic acid, alginate, or natural polymers thereof. Examplesof synthetic polymers include, without limitation, acrylics, nylons,silicones, spandex, viscose rayon, polycarboxylic acids, polyvinylacetate, polyacrylamide, polyacrylate, polyethylene glycol,polyurethanes, polylactic acid, silica, polystyrene, polyacrylonitrile,polybutadiene, polycarbonate, polyethylene, polyethylene terephthalate,poly(chlorotrifluoroethylene), poly(ethylene oxide), poly(ethyleneterephthalate), polyethylene, polyisobutylene, poly(methylmethacrylate), poly(oxymethylene), polyformaldehyde, polypropylene,polystyrene, poly(tetrafluoroethylene), poly(vinyl acetate), poly(vinylalcohol), poly(vinyl chloride), poly(vinylidene dichloride),poly(vinylidene difluoride), poly(vinyl fluoride) and/or combinations(e.g., co-polymers) thereof. Beads can also be formed from materialsother than polymers, including for example, lipids, micelles, ceramics,glass-ceramics, material composites, metals, and/or other inorganicmaterials.

In some embodiments, a bead is a degradable bead. A degradable bead caninclude one or more species (e.g., disulfide linkers, primers, otheroligonucleotides, etc.) with a labile bond such that, when thebead/species is exposed to the appropriate stimuli, the labile bond isbroken and the bead degrades. The labile bond can be a chemical bond(e.g., covalent bond, ionic bond) or can be another type of physicalinteraction (e.g., van der Waals interactions, dipole-dipoleinteractions, etc.). In some embodiments, a crosslinker used to generatea bead can include a labile bond. Upon exposure to the appropriateconditions, the labile bond can be broken and the bead degraded. Forexample, upon exposure of a polyacrylamide gel bead including cystaminecrosslinkers to a reducing agent, the disulfide bonds of the cystaminecan be broken and the bead degraded.

Degradation can refer to the disassociation of a bound or entrainedspecies (e.g., disulfide linkers, primers, other oligonucleotides, etc.)from a bead, both with and without structurally degrading the physicalbead itself. For example, entrained species can be released from beadsthrough osmotic pressure differences due to, for example, changingchemical environments. By way of example, alteration of bead pore sizesdue to osmotic pressure differences can generally occur withoutstructural degradation of the bead itself. In some embodiments, anincrease in pore size due to osmotic swelling of a bead can permit therelease of entrained species within the bead. In some embodiments,osmotic shrinking of a bead can cause a bead to better retain anentrained species due to pore size contraction.

Any suitable agent that can degrade beads can be used. In someembodiments, changes in temperature or pH can be used to degradethermo-sensitive or pH-sensitive bonds within beads. In someembodiments, chemical degrading agents can be used to degrade chemicalbonds within beads by oxidation, reduction or other chemical changes.For example, a chemical degrading agent can be a reducing agent, such asDTT, where DTT can degrade the disulfide bonds formed between acrosslinker and gel precursors, thus degrading the bead. In someembodiments, a reducing agent can be added to degrade the bead, whichcan cause the bead to release its contents. Examples of reducing agentscan include, without limitation, dithiothreitol (DTT),β-mercaptoethanol, (2S)-2-amino-1,4-dimercaptobutane (dithiobutylamineor DTBA), tris(2-carboxyethyl) phosphine (TCEP), or combinationsthereof.

Any of a variety of chemical agents can be used to trigger thedegradation of beads. Examples of chemical agents include, but are notlimited to, pH-mediated changes to the integrity of a component withinthe bead, degradation of a component of a bead via cleavage ofcross-linked bonds, and depolymerization of a component of a bead.

In some embodiments, a bead can be formed from materials that includedegradable chemical crosslinkers, such as N,N′-bis-(acryloyl)cystamine(BAC) or cystamine. Degradation of such degradable crosslinkers can beaccomplished through any variety of mechanisms. In some examples, a beadcan be contacted with a chemical degrading agent that can induceoxidation, reduction or other chemical changes. For example, a chemicaldegrading agent can be a reducing agent, such as dithiothreitol (DTT).Additional examples of reducing agents can include β-mercaptoethanol,(2S)-2-amino-1,4-dimercaptobutane (dithiobutylamine or DTBA),tris(2-carboxyethyl) phosphine (TCEP), or combinations thereof.

In some embodiments, exposure to an aqueous solution, such as water, cantrigger hydrolytic degradation, and thus degradation of the bead. Beadscan also be induced to release their contents upon the application of athermal stimulus. A change in temperature can cause a variety of changesto a bead. For example, heat can cause a solid bead to liquefy. A changein heat can cause melting of a bead such that a portion of the beaddegrades. In some embodiments, heat can increase the internal pressureof the bead components such that the bead ruptures or explodes. Heat canalso act upon heat-sensitive polymers used as materials to constructbeads.

Where degradable beads are used, it can be beneficial to avoid exposingsuch beads to the stimulus or stimuli that cause such degradation priorto a given time, in order to, for example, avoid premature beaddegradation and issues that arise from such degradation, including forexample poor flow characteristics and aggregation. By way of example,where beads include reducible cross-linking groups, such as disulfidegroups, it will be desirable to avoid contacting such beads withreducing agents, e.g., DTT or other disulfide cleaving reagents. In suchembodiments, treatment of the beads described herein will, in someembodiments be provided free of reducing agents, such as DTT. Becausereducing agents are often provided in commercial enzyme preparations, itcan be desirable to provide reducing agent free (or DTT free) enzymepreparations in treating the beads described herein. Examples of suchenzymes include, e.g., polymerase enzyme preparations, reversetranscriptase enzyme preparations, ligase enzyme preparations, as wellas many other enzyme preparations that can be used to treat the beadsdescribed herein. The terms “reducing agent free” or “DTT free”preparations refer to a preparation having less than about ⅒th, lessthan about 1/50th, or less than about 1/100th of the lower ranges forsuch materials used in degrading the beads. For example, for DTT, thereducing agent free preparation can have less than about 0.01 millimolar(mM), 0.005 mM, 0.001 mM DTT, 0.0005 mM DTT, or less than about 0.0001mM DTT. In some embodiments, the amount of DTT can be undetectable.

A degradable bead can be useful to more quickly release an attachedcapture probe (e.g., a nucleic acid molecule, a spatial barcodesequence, and/or a primer) from the bead when the appropriate stimulusis applied to the bead as compared to a bead that does not degrade. Forexample, for a species bound to an inner surface of a porous bead or inthe case of an encapsulated species, the species can have greatermobility and accessibility to other species in solution upon degradationof the bead. In some embodiments, a species can also be attached to adegradable bead via a degradable linker (e.g., disulfide linker). Thedegradable linker can respond to the same stimuli as the degradable beador the two degradable species can respond to different stimuli. Forexample, a capture probe having one or more spatial barcodes can beattached, via a disulfide bond, to a polyacrylamide bead includingcystamine. Upon exposure of the spatially barcoded bead to a reducingagent, the bead degrades and the capture probe having the one or morespatial barcode sequences is released upon breakage of both thedisulfide linkage between the capture probe and the bead and thedisulfide linkages of the cystamine in the bead.

The addition of multiple types of labile bonds to a bead can result inthe generation of a bead capable of responding to varied stimuli. Eachtype of labile bond can be sensitive to an associated stimulus (e.g.,chemical stimulus, light, temperature, pH, enzymes, etc.) such thatrelease of reagents attached to a bead via each labile bond can becontrolled by the application of the appropriate stimulus. Somenon-limiting examples of labile bonds that can be coupled to a precursoror bead include an ester linkage (e.g., cleavable with an acid, a base,or hydroxylamine), a vicinal diol linkage (e.g., cleavable via sodiumperiodate), a Diels-Alder linkage (e.g., cleavable via heat), a sulfonelinkage (e.g., cleavable via a base), a silyl ether linkage (e.g.,cleavable via an acid), a glycosidic linkage (e.g., cleavable via anamylase), a peptide linkage (e.g., cleavable via a protease), or aphosphodiester linkage (e.g., cleavable via a nuclease (e.g., DNAase)).A bond can be cleavable via other nucleic acid molecule targetingenzymes, such as restriction enzymes (e.g., restriction endonucleases).Such functionality can be useful in controlled release of reagents froma bead. In some embodiments, another reagent including a labile bond canbe linked to a bead after gel bead formation via, for example, anactivated functional group of the bead as described above. In someembodiments, a gel bead including a labile bond is reversible. In someembodiments, a gel bead with a reversible labile bond is used to captureone or more regions of interest of a biological sample. For example,without limitation, a bead including a thermolabile bond can be heatedby a light source (e.g., a laser) that causes a change in the gel beadthat facilitates capture of a biological sample in contact with the gelbead. Capture probes having one or more spatial barcodes that arereleasably, cleavably, or reversibly attached to the beads describedherein include capture probes that are released or releasable throughcleavage of a linkage between the capture probe and the bead, or thatare released through degradation of the underlying bead itself, allowingthe capture probes having the one or more spatial barcodes to beaccessed or become accessible by other reagents, or both.

Beads can have different physical properties. Physical properties ofbeads can be used to characterize the beads. Non-limiting examples ofphysical properties of beads that can differ include size, shape,circularity, density, symmetry, and hardness. For example, beads can beof different sizes. Different sizes of beads can be obtained by usingmicrofluidic channel networks configured to provide specific sized beads(e.g., based on channel sizes, flow rates, etc.). In some embodiments,beads have different hardness values that can be obtained by varying theconcentration of polymer used to generate the beads. In someembodiments, a spatial barcode attached to a bead can be made opticallydetectable using a physical property of the capture probe. For example,a nucleic acid origami, such as a deoxyribonucleic acid (DNA) origami,can be used to generate an optically detectable spatial barcode. To doso, a nucleic acid molecule, or a plurality of nucleic acid molecules,can be folded to create two-and/or three-dimensional geometric shapes.The different geometric shapes can be optically detected.

In some embodiments, special types of nanoparticles with more than onedistinct physical property can be used to make the beads physicallydistinguishable. For example, Janus particles with both hydrophilic andhydrophobic surfaces can be used to provide unique physical properties.

In some embodiments, a bead is able to identify multiple analytes (e.g.,nucleic acids, proteins, chromatin, metabolites, drugs, gRNA, andlipids) from a single cell. In some embodiments, a bead is able toidentify a single analyte from a single cell (e.g., mRNA).

A bead can have a tunable pore size. The pore size can be chosen to, forinstance, retain denatured nucleic acids. The pore size can be chosen tomaintain diffusive permeability to exogenous chemicals such as sodiumhydroxide (NaOH) and/or endogenous chemicals such as inhibitors. A beadcan be formed of a biocompatible and/or biochemically compatiblematerial, and/or a material that maintains or enhances cell viability. Abead can be formed from a material that can be depolymerized thermally,chemically, enzymatically, and/or optically.

In some embodiments, beads can be non-covalently loaded with one or morereagents. The beads can be non-covalently loaded by, for instance,subjecting the beads to conditions sufficient to swell the beads,allowing sufficient time for the reagents to diffuse into the interiorsof the beads, and subjecting the beads to conditions sufficient tode-swell the beads. Swelling of the beads can be accomplished, forinstance, by placing the beads in a thermodynamically favorable solvent,subjecting the beads to a higher or lower temperature, subjecting thebeads to a higher or lower ion concentration, and/or subjecting thebeads to an electric field.

The swelling of the beads can be accomplished by various swellingmethods. In some embodiments, swelling is reversible (e.g., bysubjecting beads to conditions that promote de-swelling). In someembodiments, the de-swelling of the beads is accomplished, for instance,by transferring the beads in a thermodynamically unfavorable solvent,subjecting the beads to lower or higher temperatures, subjecting thebeads to a lower or higher ion concentration, and/or adding or removingan electric field. The de-swelling of the beads can be accomplished byvarious de-swelling methods. In some embodiments, de-swelling isreversible (e.g., subject beads to conditions that promote swelling). Insome embodiments, the de-swelling of beads can include transferring thebeads to cause pores in the bead to shrink. The shrinking can thenhinder reagents within the beads from diffusing out of the interiors ofthe beads. The hindrance created can be due to steric interactionsbetween the reagents and the interiors of the beads. The transfer can beaccomplished microfluidically. For instance, the transfer can beachieved by moving the beads from one co-flowing solvent stream to adifferent co-flowing solvent stream. The swellability and/or pore sizeof the beads can be adjusted by changing the polymer composition of thebead.

A bead can include a polymer that is responsive to temperature so thatwhen the bead is heated or cooled, the characteristics or dimensions ofthe bead can change. For example, a polymer can includepoly(N-isopropylacrylamide). A gel bead can includepoly(N-isopropylacrylamide) and when heated the gel bead can decrease inone or more dimensions (e.g., a cross-sectional diameter, multiplecross-sectional diameters). A temperature sufficient for changing one ormore characteristics of the gel bead can be, for example, at least about0° C. (°C), 1° C., 2° C., 3° C., 4° C., 5° C., 10° C., or higher. Forexample, the temperature can be about 4° C. In some embodiments, atemperature sufficient for changing one or more characteristics of thegel bead can be, for example, at least about 25° C., 30° C., 35° C., 37°C., 40° C., 45° C., 50° C., or higher. For example, the temperature canbe about 37° C.

Functionalization of beads for attachment of capture probes can beachieved through a wide range of different approaches, including,without limitation, activation of chemical groups within a polymer,incorporation of active or activatable functional groups in the polymerstructure, or attachment at the pre-polymer or monomer stage in beadproduction. The bead can be functionalized to bind to targeted analytes,such as nucleic acids, proteins, carbohydrates, lipids, metabolites,peptides, or other analytes.

In some embodiments, a bead can contain molecular precursors (e.g.,monomers or polymers), which can form a polymer network viapolymerization of the molecular precursors. In some embodiments, aprecursor can be an already polymerized species capable of undergoingfurther polymerization via, for example, a chemical cross-linkage. Insome embodiments, a precursor can include one or more of an acrylamideor a methacrylamide monomer, oligomer, or polymer. In some embodiments,the bead can include prepolymers, which are oligomers capable of furtherpolymerization. For example, polyurethane beads can be prepared usingprepolymers. In some embodiments, a bead can contain individual polymersthat can be further polymerized together (e.g., to form a co-polymer).In some embodiments, a bead can be generated via polymerization ofdifferent precursors, such that they include mixed polymers,co-polymers, and/or block co-polymers. In some embodiments, a bead caninclude covalent or ionic bonds between polymeric precursors (e.g.,monomers, oligomers, and linear polymers), nucleic acid molecules (e.g.,oligonucleotides), primers, and other entities. In some embodiments,covalent bonds can be carbon-carbon bonds or thioether bonds.

Cross-linking of polymers can be permanent or reversible, depending uponthe particular cross-linker used. Reversible cross-linking can allow thepolymer to linearize or dissociate under appropriate conditions. In someembodiments, reversible cross-linking can also allow for reversibleattachment of a material bound to the surface of a bead. In someembodiments, a cross-linker can form a disulfide linkage. In someembodiments, a chemical cross-linker forming a disulfide linkage can becystamine or a modified cystamine.

For example, where the polymer precursor material includes a linearpolymer material, such as a linear polyacrylamide, PEG, or other linearpolymeric material, the activation agent can include a cross-linkingagent, or a chemical that activates a cross-linking agent within formeddroplets. Likewise, for polymer precursors that include polymerizablemonomers, the activation agent can include a polymerization initiator.For example, in certain embodiments, where the polymer precursorincludes a mixture of acrylamide monomer with a N,N′-bis-(acryloyl)cystamine (BAC) comonomer, an agent such astetraethylmethylenediamine (TEMED) can be provided, which can initiatethe copolymerization of the acrylamide and BAC into a cross-linkedpolymer network, or other conditions sufficient to polymerize or gel theprecursors. The conditions sufficient to polymerize or gel theprecursors can include exposure to heating, cooling, electromagneticradiation, and/or light.

Following polymerization or gelling, a polymer or gel can be formed. Thepolymer or gel can be diffusively permeable to chemical or biochemicalreagents. The polymer or gel can be diffusively impermeable tomacromolecular constituents. The polymer or gel can include one or moreof disulfide cross-linked polyacrylamide, agarose, alginate, polyvinylalcohol, polyethylene glycol (PEG)-diacrylate, PEG-acrylate, PEG-thiol,PEG-azide, PEG-alkyne, other acrylates, chitosan, hyaluronic acid,collagen, fibrin, gelatin, or elastin. The polymer or gel can includeany other polymer or gel.

In some embodiments, disulfide linkages can be formed between molecularprecursor units (e.g., monomers, oligomers, or linear polymers) orprecursors incorporated into a bead and nucleic acid molecules (e.g.,oligonucleotides, capture probes). Cystamine (including modifiedcystamines), for example, is an organic agent including a disulfide bondthat can be used as a crosslinker agent between individual monomeric orpolymeric precursors of a bead. Polyacrylamide can be polymerized in thepresence of cystamine or a species including cystamine (e.g., a modifiedcystamine) to generate polyacrylamide gel beads including disulfidelinkages (e.g., chemically degradable beads includingchemically-reducible cross-linkers). The disulfide linkages can permitthe bead to be degraded (or dissolved) upon exposure of the bead to areducing agent.

In some embodiments, chitosan, a linear polysaccharide polymer, can becross-linked with glutaraldehyde via hydrophilic chains to form a bead.Crosslinking of chitosan polymers can be achieved by chemical reactionsthat are initiated by heat, pressure, change in pH, and/or radiation.

In some embodiments, a bead can include an acrydite moiety, which incertain aspects can be used to attach one or more capture probes to thebead. In some embodiments, an acrydite moiety can refer to an acryditeanalogue generated from the reaction of acrydite with one or morespecies (e.g., disulfide linkers, primers, other oligonucleotides,etc.), such as, without limitation, the reaction of acrydite with othermonomers and cross-linkers during a polymerization reaction. Acryditemoieties can be modified to form chemical bonds with a species to beattached, such as a capture probe. Acrydite moieties can be modifiedwith thiol groups capable of forming a disulfide bond or can be modifiedwith groups already including a disulfide bond. The thiol or disulfide(via disulfide exchange) can be used as an anchor point for a species tobe attached or another part of the acrydite moiety can be used forattachment. In some embodiments, attachment can be reversible, such thatwhen the disulfide bond is broken (e.g., in the presence of a reducingagent), the attached species is released from the bead. In someembodiments, an acrydite moiety can include a reactive hydroxyl groupthat can be used for attachment of species.

In some embodiments, precursors (e.g., monomers or cross-linkers) thatare polymerized to form a bead can include acrydite moieties, such thatwhen a bead is generated, the bead also includes acrydite moieties. Theacrydite moieties can be attached to a nucleic acid molecule (e.g., anoligonucleotide), which can include a priming sequence (e.g., a primerfor amplifying target nucleic acids, random primer, primer sequence formessenger RNA) and/or one or more capture probes. The one or morecapture probes can include sequences that are the same for all captureprobes coupled to a given bead and/or sequences that are differentacross all capture probes coupled to the given bead. The capture probecan be incorporated into the bead. In some embodiments, the captureprobe can be incorporated or attached to the bead such that the captureprobe retains a free 3′ end. In some embodiments, the capture probe canbe incorporated or attached to the bead such that the capture proberetains a free 5′ end. In some embodiments, beads can be functionalizedsuch that each bead contains a plurality of different capture probes.For example, a bead can include a plurality of capture probes e.g.,Capture Probe 1, Capture Probe 2, and Capture Probe 3, and each ofCapture Probes 1, Capture Probes 2, and Capture Probes 3 contain adistinct capture domain (e.g., capture domain of Capture Probe 1includes a poly(dT) capture domain, capture domain of Capture Probe 2includes a gene-specific capture domain, and capture domain of CaptureProbe 3 includes a CRISPR-specific capture domain). By functionalizingbeads to contain a plurality of different capture domains per bead, thelevel of multiplex capability for analyte detection can be improved.

In some embodiments, precursors (e.g., monomers or cross-linkers) thatare polymerized to form a bead can include a functional group that isreactive or capable of being activated such that when it becomesreactive it can be polymerized with other precursors to generate beadsincluding the activated or activatable functional group. The functionalgroup can then be used to attach additional species (e.g., disulfidelinkers, primers, other oligonucleotides, etc.) to the beads. Forexample, some precursors including a carboxylic acid (COOH) group cancopolymerize with other precursors to form a bead that also includes aCOOH functional group. In some embodiments, acrylic acid (a speciesincluding free COOH groups), acrylamide, and bis(acryloyl)cystamine canbe co-polymerized together to generate a bead including free COOHgroups. The COOH groups of the bead can be activated (e.g., via1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) andN-Hydroxysuccinimide (NHS) or4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride(DMTMM)) such that they are reactive (e.g., reactive to amine functionalgroups where EDC/NHS or DMTMM are used for activation). The activatedCOOH groups can then react with an appropriate species (e.g., a speciesincluding an amine functional group where the carboxylic acid groups areactivated to be reactive with an amine functional group) as a functionalgroup on a moiety to be linked to the bead.

Beads including disulfide linkages in their polymeric network can befunctionalized with additional species (e.g., disulfide linkers,primers, other oligonucleotides, etc.) via reduction of some of thedisulfide linkages to free thiols. The disulfide linkages can be reducedvia, for example, the action of a reducing agent (e.g., DTT, TCEP, etc.)to generate free thiol groups, without dissolution of the bead. Freethiols of the beads can then react with free thiols of a species or aspecies including another disulfide bond (e.g., via thiol-disulfideexchange) such that the species can be linked to the beads (e.g., via agenerated disulfide bond). In some embodiments, free thiols of the beadscan react with any other suitable group. For example, free thiols of thebeads can react with species including an acrydite moiety. The freethiol groups of the beads can react with the acrydite via Michaeladdition chemistry, such that the species including the acrydite islinked to the bead. In some embodiments, uncontrolled reactions can beprevented by inclusion of a thiol capping agent such asN-ethylmalieamide or iodoacetate.

Activation of disulfide linkages within a bead can be controlled suchthat only a small number of disulfide linkages are activated. Controlcan be exerted, for example, by controlling the concentration of areducing agent used to generate free thiol groups and/or concentrationof reagents used to form disulfide bonds in bead polymerization. In someembodiments, a low concentration of reducing agent (e.g., molecules ofreducing agent:gel bead ratios) of less than or equal to about1:100,000,000,000, less than or equal to about 1:10,000,000,000, lessthan or equal to about 1:1,000,000,000, less than or equal to about1:100,000,000, less than or equal to about 1:10,000,000, less than orequal to about 1:1,000,000, less than or equal to about 1:100,000, orless than or equal to about 1:10,000) can be used for reduction.Controlling the number of disulfide linkages that are reduced to freethiols can be useful in ensuring bead structural integrity duringfunctionalization. In some embodiments, optically-active agents, such asfluorescent dyes can be coupled to beads via free thiol groups of thebeads and used to quantify the number of free thiols present in a beadand/or track a bead.

In some embodiments, addition of moieties to a bead after bead formationcan be advantageous. For example, addition of a capture probe after beadformation can avoid loss of the species (e.g., disulfide linkers,primers, other oligonucleotides, etc.) during chain transfer terminationthat can occur during polymerization. In some embodiments, smallerprecursors (e.g., monomers or cross linkers that do not include sidechain groups and linked moieties) can be used for polymerization and canbe minimally hindered from growing chain ends due to viscous effects. Insome embodiments, functionalization after bead synthesis can minimizeexposure of species (e.g., oligonucleotides) to be loaded withpotentially damaging agents (e.g., free radicals) and/or chemicalenvironments. In some embodiments, the generated hydrogel can possess anupper critical solution temperature (UCST) that can permit temperaturedriven swelling and collapse of a bead. Such functionality can aid inoligonucleotide (e.g., a primer) infiltration into the bead duringsubsequent functionalization of the bead with the oligonucleotide.Post-production functionalization can also be useful in controllingloading ratios of species in beads, such that, for example, thevariability in loading ratio is minimized. Species loading can also beperformed in a batch process such that a plurality of beads can befunctionalized with the species in a single batch.

Reagents can be encapsulated in beads during bead generation (e.g.,during polymerization of precursors). Such reagents can or cannotparticipate in polymerization. Such reagents can be entered intopolymerization reaction mixtures such that generated beads include thereagents upon bead formation. In some embodiments, such reagents can beadded to the beads after formation. Such reagents can include, forexample, capture probes (e.g., oligonucleotides), reagents for a nucleicacid amplification reaction (e.g., primers, polymerases, dNTPs,co-factors (e.g., ionic co-factors), buffers) including those describedherein, reagents for enzymatic reactions (e.g., enzymes, co-factors,substrates, buffers), reagents for nucleic acid modification reactionssuch as polymerization, ligation, or digestion, and/or reagents fortemplate preparation (e.g., tagmentation) for one or more sequencingplatforms (e.g., Nextera® for Illumina®). Such reagents can include oneor more enzymes described herein, including without limitation,polymerase, reverse transcriptase, restriction enzymes (e.g.,endonuclease), transposase, ligase, proteinase K, DNAse, etc. Suchreagents can also or alternatively include one or more reagents such aslysis agents, inhibitors, inactivating agents, chelating agents,stimulus agents. Trapping of such reagents can be controlled by thepolymer network density generated during polymerization of precursors,control of ionic charge within the bead (e.g., via ionic species linkedto polymerized species), or by the release of other species.Encapsulated reagents can be released from a bead upon bead degradationand/or by application of a stimulus capable of releasing the reagentsfrom the bead.

In some embodiments, the beads can also include (e.g., encapsulate orhave attached thereto) a plurality of capture probes that includespatial barcodes, and the optical properties of the spatial barcodes canbe used for optical detection of the beads. For example, the absorbanceof light by the spatial barcodes can be used to distinguish the beadsfrom one another. In some embodiments, a detectable label can directlyor indirectly attach to a spatial barcode and provide optical detectionof the bead. In some embodiments, each bead in a group of one or morebeads has a unique detectable label, and detection of the uniquedetectable label determines the location of the spatial barcode sequenceassociated with the bead.

Optical properties giving rise to optical detection of beads can be dueto optical properties of the bead surface (e.g., a detectable labelattached to the bead or the size of the bead), or optical propertiesfrom the bulk region of the bead (e.g., a detectable label incorporatedduring bead formation or an optical property of the bead itself). Insome embodiments, a detectable label can be associated with a bead orone or more moieties coupled to the bead.

In some embodiments, the beads include a plurality of detectable labels.For example, a fluorescent dye can be attached to the surface of thebeads and/or can be incorporated into the beads. Different intensitiesof the different fluorescent dyes can be used to increase the number ofoptical combinations that can be used to differentiate between beads.For example, if N is the number of fluorescent dyes (e.g., between 2 and10 fluorescent dyes, such as 4 fluorescent dyes) and M is the possibleintensities for the dyes (e.g., between 2 and 50 intensities, such as 20intensities), then M^(N) are the possible distinct optical combinations.In one example, 4 fluorescent dyes with 20 possible intensities can beused to generate 160,000 distinct optical combinations.

One or more optical properties of the beads or biological contents, suchas cells or nuclei, can be used to distinguish the individual beads orbiological contents from other beads or biological contents. In someembodiments, the beads are made optically detectable by including adetectable label having optical properties to distinguish the beads fromone another.

In some embodiments, optical properties of the beads can be used foroptical detection of the beads. For example, without limitation, opticalproperties can include absorbance, birefringence, color, fluorescence,luminosity, photosensitivity, reflectivity, refractive index,scattering, or transmittance. For example, beads can have differentbirefringence values based on degree of polymerization, chain length, ormonomer chemistry.

In some embodiments, nanobeads, such as quantum dots or Janus beads, canbe used as optical labels or components thereof. For example, a quantumdot can be attached to a spatial barcode of a bead.

Optical labels of beads can provide enhanced spectral resolution todistinguish between beads with unique spatial barcodes (e.g., beadsincluding unique spatial barcode sequences). In some embodiments, afirst bead includes a first optical label and spatial barcodes eachhaving a first spatial barcode sequence. A second bead includes a secondoptical label and spatial barcodes each having a second spatial barcodesequence. The first optical label and second optical label can bedifferent (e.g., provided by two different fluorescent dyes or the samefluorescent dye at two different intensities). The first and secondspatial barcode sequences can be different nucleic acid sequences. Insome embodiments, the beads can be imaged to identify the first andsecond optical labels, and the first and second optical barcodes canthen be used to associate the first and second optical labels with thefirst and second spatial barcode sequences, respectively.

Optical labels can be included while generating the beads. For example,optical labels can be included in the polymer structure of a gel bead,or attached at the pre-polymer or monomer stage in bead production. Insome embodiments, the beads include moieties that attach to one or moreoptical labels (e.g., at a surface of a bead and/or within a bead). Insome embodiments, optical labels can be loaded into the beads with oneor more reagents. For example, reagents and optical labels can be loadedinto the beads by diffusion of the reagents (e.g., a solution ofreagents including the optical barcodes). In some embodiments, opticallabels can be included while preparing spatial barcodes. For example,spatial barcodes can be prepared by synthesizing molecules includingbarcode sequences (e.g., using a split pool or combinatorial approach).Optical labels can be attached to spatial barcodes prior to attachingthe spatial barcodes to a bead. In some embodiments, optical labels canbe included after attaching spatial barcodes to a bead. For example,optical labels can be attached to spatial barcodes coupled to the bead.In some embodiments, spatial barcodes or sequences thereof can bereleasably or cleavably attached to the bead. Optical labels can bereleasably or non-releasably attached to the bead. In some embodiments,a first bead (e.g., a bead including a plurality of spatial barcodes)can be coupled to a second bead including one or more optical labels.For example, the first bead can be covalently coupled to the second beadvia a chemical bond. In some embodiments, the first bead can benon-covalently associated with the second bead.

The first and/or second bead can include a plurality of spatialbarcodes. The plurality of spatial barcodes coupled to a given bead caninclude the same barcode sequences. Where both the first and secondbeads include spatial barcodes, the first and second beads can includespatial barcodes including the same barcode sequences or differentbarcode sequences.

Bead arrays containing captured analytes can be processed in bulk orpartitioned into droplet emulsions for preparing sequencing libraries.In some embodiments, next generation sequencing reads are clustered andcorrelated to the spatial position of the spatial barcode on the beadarray. For example, the information can be computationally superimposedover a high-resolution image of the tissue section to identify thelocation(s), where the analytes were detected.

In some embodiments, de-cross linking can be performed to account forde-crosslinking chemistries that may be incompatible with certainbarcoding/library prep biochemistry (e.g., presence of proteases). Forexample, a two-step process is possible. In the first step, beads can beprovided in droplets such that DNA binds to the beads after theconventional de-crosslinking chemistry is performed. In the second step,the emulsion is broken and beads collected and then re-encapsulatedafter washing for further processing.

In some embodiments, beads can be affixed or attached to a substrateusing photochemical methods. For example, a bead can be functionalizedwith perfluorophenylazide silane (PFPA silane), contacted with asubstrate, and then exposed to irradiation (see, e.g., Liu et al. (2006)Journal of the American Chemical Society 128, 14067-14072). For example,immobilization of antraquinone-functionalized substrates (see, e.g.,Koch et al. (2000) Bioconjugate Chem. 11, 474-483, the entire contentsof which are herein incorporated by reference).

The arrays can also be prepared by bead self-assembly. Each bead can becovered with hundreds of thousands of copies of a specificoligonucleotide. In some embodiments, each bead can be covered withabout 1,000 to about 1,000,000 oligonucleotides. In some embodiments,each bead can be covered with about 1,000,000 to about 10,000,000oligonucleotides. In some embodiments, each bead can covered with about2,000,000 to about 3,000,000, about 3,000,000 to about 4,000,000, about4,000,000 to about 5,000,000, about 5,000,000 to about 6,000,000, about6,000,000 to about 7,000,000, about 7,000,000 to about 8,000,000, about8,000,000 to about 9,000,000, or about 9,000,000 to about 10,000,000oligonucleotides. In some embodiments, each bead can be covered withabout 10,000,000 to about 100,000,000 oligonucleotides. In someembodiments, each bead can be covered with about 100,000,000 to about1,000,000,000 oligonucleotides. In some embodiments, each bead can becovered with about 1,000,000,000 to about 10,000,000,000oligonucleotides. The beads can be irregularly distributed across etchedsubstrates during the array production process. During this process, thebeads can be self-assembled into arrays (e.g., on a fiber-optic bundlesubstrate or a silica slide substrate). In some embodiments, the beadsirregularly arrive at their final location on the array. Thus, the beadlocation may need to be mapped or the oligonucleotides may need to besynthesized based on a predetermined pattern.

Beads can be affixed or attached to a substrate covalently,non-covalently, with adhesive, or a combination thereof. The attachedbeads can be, for example, layered in a monolayer, a bilayer, atrilayer, or as a cluster. As defined herein, a “monolayer” generallyrefers to an arrayed series of probes, beads, spots, dots, features,micro-locations, or islands that are affixed or attached to a substrate,such that the beads are arranged as one layer of single beads. In someembodiments, the beads are closely packed.

As defined herein, the phrase “substantial monolayer” or “substantiallyform(s) a monolayer” generally refers to (the formation of) an arrayedseries of probes, beads, microspheres, spots, dots, features,micro-locations, or islands that are affixed or attached to a substrate,such that about 50% to about 99% (e.g., about 50% to about 98%) of thebeads are arranged as one layer of single beads. This arrangement can bedetermined using a variety of methods, including microscopic imaging.

In some embodiments, the monolayer of beads is a located in a predefinedarea on the substrate. For example, the predefined area can bepartitioned with physical barriers, a photomask, divots in thesubstrate, or wells in the substrate.

As used herein, the term “reactive element” generally refers to amolecule or molecular moiety that can react with another molecule ormolecular moiety to form a covalent bond. Reactive elements include, forexample, amines, aldehydes, alkynes, azides, thiols, haloacetyls,pyridyl disulfides, hydrazides, carboxylic acids, alkoxyamines,sulfhydryls, maleimides, Michael acceptors, hydroxyls, and activeesters. Some reactive elements, for example, carboxylic acids, can betreated with one or more activating agents (e.g., acylating agents,isourea-forming agents) to increase susceptibility of the reactiveelement to nucleophilic attack. Non-limiting examples of activatingagents include N-hydroxysuccinimide, N-hydroxysulfosuccinimide,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, dicyclohexylcarbodiimide,diisopropylcarbodiiimide, 1-hydroxybenzotriazole,(benzotriazol-1-yloxy)tripyrrolidinophosphonium hexfluorophosphate,(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate,4-(N,N-dimethylamino)pyridine, and carbonyldiimidazole.

In some embodiments, the reactive element is bound directly to a bead.For example, hydrogel beads can be treated with an acrylic acid monomerto form acrylic acid-functionalized hydrogel beads. In some cases, thereactive element is bound indirectly to the bead via one or morelinkers. As used herein, a “linker” generally refers to amultifunctional (e.g., bifunctional, trifunctional) reagent used forconjugating two or more chemical moieties. A linker can be a cleavablelinker that can undergo induced dissociation. For example, thedissociation can be induced by a solvent (e.g., hydrolysis andsolvolysis); by irradiation (e.g., photolysis); by an enzyme (e.g.,enzymolysis); or by treatment with a solution of specific pH (e.g., pH4, 5, 6, 7, or 8).

In some embodiments, the reactive element is bound directly to asubstrate. For example, a glass slide can be coated with(3-aminopropyl)triethoxysilane. In some embodiments, the reactiveelement is bound indirectly to a substrate via one or more linkers.

Methods for Covalently Bonding Beads to a Substrate

Provided herein are methods for the covalent bonding of beads (e.g.,optically labeled beads, hydrogel beads, microsphere beads) to asubstrate.

In some embodiments, the beads are coupled to a substrate via a covalentbond between a first reactive element and a second reactive element. Insome embodiments, the covalently-bound beads substantially form amonolayer of beads (e.g., hydrogel beads, microsphere beads) on thesubstrate.

In some embodiments, the beads are functionalized with a first reactiveelement, which is directly bound to the beads. In some embodiments, thebeads are functionalized with a first reactive element, which isindirectly bound to the beads via a linker. In some embodiments, thelinker is a benzophenone. In some embodiments, the linker is an aminomethacrylamide. For example, the linker can be 3-aminopropylmethacrylamide. In some embodiments, the linker is a PEG linker. In someembodiments, the linker is a cleavable linker.

In some embodiments, the substrate is functionalized with a secondreactive element, which is directly bound to the substrate. In someembodiments, the substrate is functionalized with a second reactiveelement, which is indirectly bound to the beads via a linker. In someembodiments, the linker is a benzophenone. For example, the linker canbe benzophenone. In some embodiments, the linker is an aminomethacrylamide. For example, the linker can be 3-aminopropylmethacrylamide. In some embodiments, the linker is a PEG linker. In someembodiments, the linker is a cleavable linker.

In some embodiments, the substrate is a glass slide. In someembodiments, the substrate is a pre-functionalized glass slide.

In some embodiments, about 99% of the covalently-bound beads form amonolayer of beads on the substrate. In some embodiments, about 50% toabout 98% form a monolayer of beads on the substrate. For example, about50% to about 95%, about 50% to about 90%, about 50% to about 85%, about50% to about 80%, about 50% to about 75%, about 50% to about 70%, about50% to about 65%, about 50% to about 60%, or about 50% to about 55% ofthe covalently-bound beads form a monolayer of beads on the substrate.In some embodiments, about 55% to about 98%, about 60% to about 98%,about 65% to about 98%, about 70% to about 98%, about 75% to about 98%,about 80% to about 98%, about 85% to about 98%, about 90% to about 95%,or about 95% to about 98% of the covalently-bound beads form a monolayerof beads on the substrate. In some embodiments, about 55% to about 95%,about 60% to about 90%, about 65% to about 95%, about 70% to about 95%,about 75% to about 90%, about 75% to about 95%, about 80% to about 90%,about 80% to about 95%, about 85% to about 90%, or about 85% to about95% of the covalently-bound beads for a monolayer of beads on thesubstrate.

In some embodiments, at least one of the first reactive element and thesecond reactive element is selected from the group consisting of:

wherein

-   R¹ is selected from H, C₁-C₆ alkyl, or —SO₃;-   R² is C₁-C₆ alkyl; and-   X is a halo moiety.

In some embodiments, at least one of the first reactive element or thesecond reactive element comprises

wherein the

indicates the point of attachment of the first reactive element or thesecond reactive element to the bead (e.g., hydrogel bead or microspherebead) or to the substrate.

In some embodiments, at least one of the first reactive element or thesecond reactive element is selected from the group consisting of:

wherein

-   R¹ is selected from H, C₁-C₆ alkyl, or —SO₃;-   R² is C₁-C₆ alkyl; and-   X is a halo moiety.

In some embodiments, at least one of the first reactive element or thesecond reactive element comprises

wherein R¹ is selected from H, C₁-C₆ alkyl, or —SO₃. In someembodiments, R¹ is H. In some embodiments, R¹ is C₁-C₆ alkyl. In someembodiments, R¹ is —SO₃.

In some embodiments, at least one of the first reactive element or thesecond reactive element comprises

wherein R² is C₁-C₆ alkyl. In some embodiments, R² is methyl.

In some embodiments, at least one of the first reactive element or thesecond reactive element comprises

Is some embodiments,

can be reacted with an activating agent to form an active ester. In someembodiments, the active ester is

In some embodiments, the activating agent is an acylating agent (e.g.,N-hydroxysuccinimide and N-hydroxysulfosuccinimide). In someembodiments, the activating agent is an O-acylisourea-forming agent(e.g., 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC),dicyclohexylcarbodiimide, and diisopropylcarbodiiimide). In someembodiments, the activating agent is a combination of at least oneacylating agent and at least one O-isourea-forming agents (e.g.,N-hydroxysuccinimide (NHS),1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC),N-hydroxysulfosuccinimide (sulfo-NHS), and a combination thereof).

In some embodiments, at least one of the first reactive element or thesecond reactive element comprises

In some embodiments, at least one of the first reactive element or thesecond reactive element comprises

wherein X is a halo moiety. For example, X is chloro, bromo, or iodo.

In some embodiments, at least one of the first reactive element or thesecond reactive element comprises

In some embodiments, at least one of the first reactive element or thesecond reactive element comprises

In some embodiments, at least one of the first reactive element or thesecond reactive element comprises

In some embodiments, at least one of the first reactive element or thesecond reactive element is selected from the group consisting of:

wherein

-   R³ is H or C₁-C₆ alkyl; and-   R⁴ is H or trimethylsilyl.

In some embodiments, at least one of the first reactive element or thesecond reactive element comprises

wherein R⁴ is H or trimethylsilyl. In some embodiments, R⁴ is H.

In some embodiments, at least one of the first reactive element or thesecond reactive element is selected from the group consisting of:

wherein R³ is H or C₁-C₆ alkyl. In some embodiments, R³ is H. In someembodiments, R³ is C₁-C₆ alkyl.

In some embodiments, at least one of the first reactive element or thesecond reactive element comprises

wherein R³ is H or C₁-C₆ alkyl. In some embodiments, R³ is H. In someembodiments, R³ is C₁-C₆ alkyl.

In some embodiments, at least one of the first reactive elements or thesecond reactive elements comprises

In some embodiments, at least one of the first reactive elements or thesecond reactive elements comprises

In some embodiments, one of the first reactive elements or the secondreactive elements is selected from the group consisting of:

wherein

-   R¹ is selected from H, C₁-C₆ alkyl, or —SO₃;

-   R² is C₁-C₆ alkyl;

-   X is a halo moiety;

-   and the other of the first reactive element or the second reactive    element is selected from the group consisting of:

-   

-   

-   

-   

-   wherein    -   R³ is H or C₁-C₆ alkyl; and    -   R⁴ is H or trimethylsilyl.

In some embodiments, one of the first reactive elements or the secondreactive elements is selected from the group consisting of

wherein R³ is H or C₁-C₆ alkyl; and the other of the first reactiveelement or the second reactive element is

wherein R⁴ is H or trimethylsilyl. In some embodiments, R³ is H. In someembodiments, R³ is C₁-C₆ alkyl. In some embodiments, R⁴ is H. In someembodiments, R⁴ is trimethylsilyl.

In some embodiments, one of the first reactive element or the secondreactive element is selected from the group consisting of:

wherein

-   R¹ is selected from H, C₁-C₆ alkyl, or —SO₃;

-   R² is C₁-C₆ alkyl;

-   X is a halo moiety;

-   and the other of the first reactive element or the second reactive    element is selected from the group consisting of:

-   

-   

-   

-   wherein R³ is H or C₁-C₆ alkyl. In some embodiments, R¹ is H. In    some embodiments, R¹ is C₁-C₆ alkyl. In some embodiments, R¹ is    —SO₃. In some embodiments, R² is methyl. In some embodiments, X is    iodo. In some embodiments, R³ is H. In some embodiments, R³ is C₁-C₆    alkyl.

In some embodiments, one of the first reactive elements or the secondreactive elements is selected from the group consisting of:

wherein

-   R¹ is selected from H, C₁-C₆ alkyl, or —SO₃;

-   R² is C₁-C₆ alkyl;

-   and the other of the first reactive elements or the second reactive    elements comprises

-   

-   wherein R³ is H or C₁-C₆ alkyl. In some embodiments, R¹ is H. In    some embodiments, R¹ is C₁-C₆ alkyl. In some embodiments, R¹ is    —SO₃. In some embodiments, R² is methyl. In some embodiments, R³    is H. In some embodiments, R³ is C₁-C₆ alkyl.

In some embodiments, one of the first reactive element or the secondreactive element is selected from the group consisting of:

-   wherein X is a halo moiety;

-   and the other of the first reactive element or the second reactive    element comprises

-   

-   some embodiments, X is bromo. In some embodiments, X is iodo.

In some embodiments, one of the first reactive element or the secondreactive element is selected from the group consisting of

and the other of the first reactive element or the second reactiveelement comprises

The term “halo” refers to fluoro (F), chloro (Cl), bromo (Br), or iodo(I).

The term “alkyl” refers to a hydrocarbon chain that may be a straightchain or branched chain, containing the indicated number of carbonatoms. For example, C₁₋₁₀ indicates that the group may have from 1 to 10(inclusive) carbon atoms in it. Non-limiting examples include methyl,ethyl, iso-propyl, tert-butyl, n-hexyl.

The term “haloalkyl” refers to an alkyl, in which one or more hydrogenatoms is/are replaced with an independently selected halo.

The term “alkoxy” refers to an -O-alkyl radical (e.g., —OCH₃).

The term “alkylene” refers to a divalent alkyl (e.g., —CH₂—).

The term “alkenyl” refers to a hydrocarbon chain that may be a straightchain or branched chain having one or more carbon-carbon double bonds.The alkenyl moiety contains the indicated number of carbon atoms. Forexample, C₂₋₆ indicates that the group may have from 2 to 6 (inclusive)carbon atoms in it.

The term “alkynyl” refers to a hydrocarbon chain that may be a straightchain or branched chain having one or more carbon-carbon triple bonds.The alkynyl moiety contains the indicated number of carbon atoms. Forexample, C₂₋₆ indicates that the group may have from 2 to 6 (inclusive)carbon atoms in it.

The term “aryl” refers to a 6-20 carbon mono-, bi-, tri- or polycyclicgroup wherein at least one ring in the system is aromatic (e.g.,6-carbon monocyclic, 10-carbon bicyclic, or 14-carbon tricyclic aromaticring system); and wherein 0, 1, 2, 3, or 4 atoms of each ring may besubstituted by a substituent. Examples of aryl groups include phenyl,naphthyl, tetrahydronaphthyl, and the like.

Methods for Non-Covalently Bonding Beads to a Substrate

Provided herein are methods for the non-covalent bonding of beads (e.g.,optically-labeled beads, hydrogel beads, or microsphere beads) to asubstrate.

In some embodiments, beads are coupled to a substrate via a non-covalentbond between a first affinity group and a second affinity group. In someembodiments, the non-covalently-bound beads substantially form amonolayer of beads (e.g., hydrogel beads, microsphere beads) on thesubstrate.

In some embodiments, the beads are functionalized with a first affinitygroup, which is directly bound to the beads. In some embodiments, thebeads are functionalized with a first affinity group, which isindirectly bound to the beads via a linker. In some embodiments, thelinker is a benzophenone. In some embodiments, the linker is an aminomethacrylamide. For example, the linker can be 3-aminopropylmethacrylamide. In some embodiments, the linker is a PEG linker. In someembodiments, the linker is a cleavable linker.

In some embodiments, the substrate is functionalized with a secondaffinity group, which is directly bound to the substrate. In someembodiments, the substrate is functionalized with a second affinitygroup, which is indirectly bound to the beads via a linker. In someembodiments, the linker is a benzophenone. In some embodiments, thelinker is an amino methacrylamide. For example, the linker can be3-aminopropyl methacrylamide. In some embodiments, the linker is a PEGlinker. In some embodiments, the linker is a cleavable linker.

In some embodiments the first affinity group or the second affinitygroup is biotin, and the other of the first affinity group or the secondaffinity group is streptavidin.

In some embodiments, about 99% of the non-covalently-bound beads form amonolayer of beads on the substrate. In some embodiments, about 50% toabout 98% form a monolayer of beads on the substrate. For example, about50% to about 95%, about 50% to about 90%, about 50% to about 85%, about50% to about 80%, about 50% to about 75%, about 50% to about 70%, about50% to about 65%, about 50% to about 60%, or about 50% to about 55% ofthe non-covalently-bound beads form a monolayer of beads on thesubstrate. In some embodiments, about 55% to about 98%, about 60% toabout 98%, about 65% to about 98%, about 70% to about 98%, about 75% toabout 98%, about 80% to about 98%, about 85% to about 98%, about 90% toabout 95%, or about 95% to about 98% of the non-covalently-bound beadsform a monolayer of beads on the substrate. In some embodiments, about55% to about 95%, about 60% to about 90%, about 65% to about 95%, about70% to about 95%, about 75% to about 90%, about 75% to about 95%, about80% to about 90%, about 80% to about 95%, about 85% to about 90%, orabout 85% to about 95% of the non-covalently-bound beads for a monolayerof beads on the substrate.

In some embodiments, the monolayer of beads is a formed in a predefinedarea on the substrate. In some embodiments, the predefined area ispartitioned with physical barriers. For example, divots or wells in thesubstrate. In some embodiments, the predefined area is partitioned usinga photomask. For example, the substrate is coated with a photo-activatedsolution, dried, then irradiated under a photomask. In some embodiments,the photo-activated solution is UV-activated.

As used herein, an “adhesive” generally refers to a substance used forsticking objects or materials together. Adhesives include, for example,glues, pastes, liquid tapes, epoxy, bioadhesives, gels, and mucilage. Insome embodiments, an adhesive is liquid tape. In some embodiments, theadhesive is glue.

In some embodiments, beads are adhered to a substrate using an adhesive(e.g., liquid tape, glue, paste). In some embodiments, the adhered beadssubstantially form a monolayer of beads on the substrate (e.g., a glassslide). In some embodiments, the beads are hydrogel beads. In someembodiments, the beads are microsphere beads. In some embodiments, thebeads are coated with the adhesive, and then the beads are contactedwith the substrate. In some embodiments, the substrate is coated withthe adhesive, and then the substrate is contacted with the beads. Insome embodiments, both the substrate is coated with the adhesive and thebeads are coated with the adhesive, and then the beads and substrate arecontacted with one another.

In some embodiments, about 99% of the adhered beads form a monolayer ofbeads on the substrate. In some embodiments, about 50% to about 98% forma monolayer of beads on the substrate. For example, about 50% to about95%, about 50% to about 90%, about 50% to about 85%, about 50% to about80%, about 50% to about 75%, about 50% to about 70%, about 50% to about65%, about 50% to about 60%, or about 50% to about 55% of the adheredbeads form a monolayer of beads on the substrate. In some embodiments,about 55% to about 98%, about 60% to about 98%, about 65% to about 98%,about 70% to about 98%, about 75% to about 98%, about 80% to about 98%,about 85% to about 98%, about 90% to about 95%, or about 95% to about98% of the adhered beads form a monolayer of beads on the substrate. Insome embodiments, about 55% to about 95%, about 60% to about 90%, about65% to about 95%, about 70% to about 95%, about 75% to about 90%, about75% to about 95%, about 80% to about 90%, about 80% to about 95%, about85% to about 90%, or about 85% to about 95% of the adhered beads for amonolayer of beads on the substrate.

In some embodiments, beads can be deposited onto a biological samplesuch that the deposited beads form a monolayer of beads on thebiological sample (e.g., over or under the biological sample). In someembodiments, beads deposited on the substrate can self-assemble into amonolayer of beads that saturate the intended surface area of thebiological sample under investigation. In this approach, bead arrays canbe designed, formulated, and prepared to evaluate a plurality ofanalytes from a biological sample of any size or dimension. In someembodiments, the concentration or density of beads (e.g., gel beads)applied to the biological sample is such that the area as a whole, orone or more regions of interest in the biological sample, is saturatedwith a monolayer of beads. In some embodiments, the beads are contactedwith the biological sample by pouring, pipetting, spraying, and thelike, onto the biological sample. Any suitable form of bead depositioncan be used.

In some embodiments, the biological sample can be confined to a specificregion or area of the array. For example, a biological sample can beaffixed to a glass slide and a chamber, gasket, or cage positioned overthe biological sample to act as a containment region or frame withinwhich the beads are deposited. As will be apparent, the density orconcentration of beads needed to saturate an area or biological samplecan be readily determined by one of ordinary skill in the art (e.g.,through microscopic visualization of the beads on the biologicalsample). In some embodiments, the bead array contains microfluidicchannels to direct reagents to the spots or beads of the array.

Feature Geometric Attributes

Features on an array can have a variety of sizes. In some embodiments, afeature of an array can have a diameter or maximum dimension between 1µm to 100 µm. For example, between 1 µm to 10 µm, 1 µm to 20 µm, 1 µm to30 µm, 1 µm to 40 µm, 1 µm to 50 µm, 1 µm to 60 µm, 1 µm to 70 µm, 1 µmto 80 µm, 1 µm to 90 µm, 90 µm to 100 µm, 80 µm to 100 µm, 70 µm to 100µm, 60 µm to 100 µm, 50 µm to 100 µm, 40 µm to 100 µm, 30 µm to 100 µm,20 µm to 100 µm, or 10 µm to 100 µm. In some embodiments, the featurehas a diameter or maximum dimension between 30 µm to 100 µm, 40 µm to 90µm, 50 µm to 80 µm, 60 µm to 70 µm, or any range within the disclosedsub-ranges. In some embodiments, the feature has a diameter or maximumdimension no larger than 95 µm, 90 µm, 85 µm, 80 µm, 75 µm, 70 µm, 65µm, 60 µm, 55 µm, 50 µm, 45 µm, 40 µm, 35 µm, 30 µm, 25 µm, 20 µm, 15µm, 14 µm, 13 µm, 12 µm, 11 µm, 10 µm, 9 µm, 8 µm, 7 µm, 6 µm, 5 µm, 4µm, 3 µm, 2 µm, or 1 µm. In some embodiments, the feature has a diameteror maximum dimension of approximately 65 µm.

In some embodiments, the size and/or shape of a plurality of features ofan array are approximately uniform. In some embodiments, the size and/orshape of a plurality of features of an array is not uniform. Forexample, in some embodiments, features in an array can have an averagecross-sectional dimension, and a distribution of cross-sectionaldimensions among the features can have a full-width and half-maximumvalue of 0% or more (e.g., 5% or more, 10% or more, 20% or more, 30% ormore, 40% or more, 50% or more, 70% or more, or 100% or more) of theaverage cross-sectional dimension for the distribution.

In certain embodiments, features in an array can have an averagecross-sectional dimension of between about 1 µm and about 10 µm. Thisrange in average feature cross-sectional dimension corresponds to theapproximate diameter of a single mammalian cell. Thus, an array of suchfeatures can be used to detect analytes at, or below, mammaliansingle-cell resolution.

In some embodiments, a plurality of features has a mean diameter or meanmaximum dimension of about 0.1 µm to about 100 µm (e.g., about 0.1 µm toabout 5 µm, about 1 µm to about 10 µm, about 1 µm to about 20 µm, about1 µm to about 30 µm, about 1 µm to about 40 µm, about 1 µm to about 50µm, about 1 µm to about 60 µm, about 1 µm to about 70 µm, about 1 µm toabout 80 µm, about 1 µm to about 90 µm, about 90 µm to about 100 µm,about 80 µm to about 100 µm, about 70 µm to about 100 µm, about 60 µm toabout 100 µm, about 50 µm to about 100 µm, about 40 µm to about 100 µm,about 30 µm to about 100 µm, about 20 µm to about 100 µm, or about 10 µmto about 100 µm). In some embodiments, the plurality of features has amean diameter or mean maximum dimension between 30 µm to 100 µm, 40 µmto 90 µm, 50 µm to 80 µm, 60 µm to 70 µm, or any range within thedisclosed sub-ranges. In some embodiments, the plurality of features hasa mean diameter or a mean maximum dimension no larger than 95 µm, 90 µm,85 µm, 80 µm, 75 µm, 70 µm, 65 µm, 60 µm, 55 µm, 50 µm, 45 µm, 40 µm, 35µm, 30 µm, 25 µm, 20 µm, 15 µm, 14 µm, 13 µm, 12 µm, 11 µm, 10 µm, 9 µm,8 µm, 7 µm, 6 µm, 5 µm, 4 µm, 3 µm, 2 µm, or 1 µm. In some embodiments,the plurality of features has a mean average diameter or a mean maximumdimension of approximately 65 µm.

In some embodiments, where the feature is a bead, the bead can have adiameter or maximum dimension no larger than 100 µm (e.g., no largerthan 95 µm, 90 µm, 85 µm, 80 µm, 75 µm, 70 µm, 65 µm, 60 µm, 55 µm, 50µm, 45 µm, 40 µm, 35 µm, 30 µm, 25 µm, 20 µm, 15 µm, 14 µm, 13 µm, 12µm, 11 µm, 10 µm, 9 µm, 8 µm, 7 µm, 6 µm, 5 µm, 4 µm, 3 µm, 2 µm, or 1µm).

In some embodiments, a plurality of beads has an average diameter nolarger than 100 µm. In some embodiments, a plurality of beads has anaverage diameter or maximum dimension no larger than 95 µm, 90 µm, 85µm, 80 µm, 75 µm, 70 µm, 65 µm, 60 µm, 55 µm, 50 µm, 45 µm, 40 µm, 35µm, 30 µm, 25 µm, 20 µm, 15 µm, 14 µm, 13 µm, 12 µm, 11 µm, 10 µm, 9 µm,8 µm, 7 µm, 6 µm, 5 µm, 4 µm, 3 µm, 2 µm, or 1 µm.

In some embodiments, the volume of the bead can be at least about 1 µm³,e.g., at least 1 µm³, 2 µm³, 3 µm³, 4 µm³, 5 µm³, 6 µm³, 7 µm³, 8 µm³, 9µm³, 10 µm³, 12 µm³, 14 µm³, 16 µm³, 18 µm³, 20 µm³, 25 µm³, 30 µm³, 35µm³, 40 µm³, 45 µm³, 50 µm³, 55 µm³, 60 µm³, 65 µm³, 70 µm³, 75 µm³, 80µm³, 85 µm³, 90 µm³, 95 µm³, 100 µm³, 125 µm³, 150 µm³, 175 µm³, 200µm³, 250 µm³, 300 µm³, 350 µm³, 400 µm³, 450 µm³, µm³, 500 µm³, 550 µm³,600 µm³, 650 µm³, 700 µm³, 750 µm³, 800 µm³, 850 µm³, 900 µm³, 950 µm³,1000 µm³, 1200 µm³, 1400 µm³, 1600 µm³, 1800 µm³, 2000 µm³, 2200 µm³,2400 µm³, 2600 µm³, 2800 µm³, 3000 µm³, or greater.

In some embodiments, the bead can have a volume of between about 1 µm³and 100 µm³, such as between about 1 µm³ and 10 µm³, between about10 µm³and 50 µm³, or between about 50 µm³ and 100 µm³. In some embodiments,the bead can include a volume of between about 100 µm³ and 1000 µm³,such as between about 100 µm³ and 500 µm³ or between about 500 µm³ and1000 µm³. In some embodiments, the bead can include a volume betweenabout 1000 µm³ and 3000 µm³, such as between about 1000 µm³ and 2000 µm³or between about 2000 µm³ and 3000 µm³. In some embodiments, the beadcan include a volume between about 1 µm³ and 3000 µm³, such as betweenabout 1 µm³ and 2000 µm³, between about 1 µm³ and 1000 µm³, betweenabout 1 µm³ and 500 µm³, or between about 1 µm³ and 250 µm³.

The bead can include one or more cross-sections that can be the same ordifferent. In some embodiments, the bead can have a first cross-sectionthat is different from a second cross-section. The bead can have a firstcross-section that is at least about 0.0001 micrometer, 0.001micrometer, 0.01 micrometer, 0.1 micrometer, or 1 micrometer. In someembodiments, the bead can include a cross-section (e.g., a firstcross-section) of at least about 1 micrometer (µm), 2 µm, 3 µm, 4 µm, 5µm, 6 µm, 7 µm, 8 µm, 9 µm, 10 µm, 11 µm, 12 µm, 13 µm, 14 µm, 15 µm, 16µm, 17 µm, 18 µm, 19 µm, 20 µm, 25 µm, 30 µm, 35 µm, 40 µm, 45 µm, 50µm, 55 µm, 60 µm, 65 µm, 70 µm, 75 µm, 80 µm, 85 µm, 90 µm, 100 µm, 120µm, 140 µm, 160 µm, 180 µm, 200 µm, 250 µm, 300 µm, 350 µm, 400 µm, 450µm, 500 µm, 550 µm, 600 µm, 650 µm, 700 µm, 750 µm, 800 µm, 850 µm, 900µm, 950 µm, 1 millimeter (mm), or greater. In some embodiments, the beadcan include a cross-section (e.g., a first cross-section) of betweenabout 1 µm and 500 µm, such as between about 1 µm and 100 µm, betweenabout 100 µm and 200 µm, between about 200 µm and 300 µm, between about300 µm and 400 µm, or between about 400 µm and 500 µm. For example, thebead can include a cross-section (e.g., a first cross-section) ofbetween about 1 µm and 100 µm. In some embodiments, the bead can have asecond cross-section that is at least about 1 µm. For example, the beadcan include a second cross-section of at least about 1 micrometer (µm),2 µm, 3 µm, 4 µm, 5 µm, 6 µm, 7 µm, 8 µm, 9 µm, 10 µm, 11 µm, 12 µm, 13µm, 14 µm, 15 µm, 16 µm, 17 µm, 18 µm, 19 µm, 20 µm, 25 µm, 30 µm, 35µm, 40 µm, 45 µm, 50 µm, 55 µm, 60 µm, 65 µm, 70 µm, 75 µm, 80 µm, 85µm, 90 µm, 100 µm, 120 µm, 140 µm, 160 µm, 180 µm, 200 µm, 250 µm, 300µm, 350 µm, 400 µm, 450 µm, 500 µm, 550 µm, 600 µm, 650 µm, 700 µm, 750µm, 800 µm, 850 µm, 900 µm, 950 µm, 1 millimeter (mm), or greater. Insome embodiments, the bead can include a second cross-section of betweenabout 1 µm and 500 µm, such as between about 1 µm and 100 µm, betweenabout 100 µm and 200 µm, between about 200 µm and 300 µm, between about300 µm and 400 µm, or between about 400 µm and 500 µm. For example, thebead can include a second cross-section of between about 1 µm and 100µm.

In some embodiments, beads can be of a nanometer scale (e.g., beads canhave a diameter or maximum cross-sectional dimension of about 100nanometers (nm) to about 900 nanometers (nm) (e.g., 850 nm or less, 800nm or less, 750 nm or less, 700 nm or less, 650 nm or less, 600 nm orless, 550 nm or less, 500 nm or less, 450 nm or less, 400 nm or less,350 nm or less, 300 nm or less, 250 nm or less, 200 nm or less, 150 nmor less). A plurality of beads can have an average diameter or averagemaximum cross-sectional dimension of about 100 nanometers (nm) to about900 nanometers (nm) (e.g., 850 nm or less, 800 nm or less, 750 nm orless, 700 nm or less, 650 nm or less, 600 nm or less, 550 nm or less,500 nm or less, 450 nm or less, 400 nm or less, 350 nm or less, 300 nmor less, 250 nm or less, 200 nm or less, 150 nm or less). In someembodiments, a bead has a diameter or size that is about the size of asingle cell (e.g., a single cell under evaluation).

Beads can be of uniform size or heterogeneous size. “Polydispersity”generally refers to heterogeneity of sizes of molecules or particles.The polydispersity (PDI) can be calculated using the equation PDI =Mw/Mn, where Mw is the weight-average molar mass and Mn is thenumber-average molar mass. In certain embodiments, beads can be providedas a population or plurality of beads having a relatively monodispersesize distribution. Where it can be desirable to provide relativelyconsistent amounts of reagents, maintaining relatively consistent beadcharacteristics, such as size, can contribute to the overallconsistency.

In some embodiments, the beads provided herein can have sizedistributions that have a coefficient of variation in theircross-sectional dimensions of less than 50%, less than 40%, less than30%, less than 20%, less than 15%, less than 10%, less than 5%, orlower. In some embodiments, a plurality of beads provided herein has apolydispersity index of less than 50%, less than 45%, less than 40%,less than 35%, less than 30%, less than 25%, less than 20%, less than15%, less than 10%, less than 5%, or lower.

Array Geometric Attributes

In some embodiments, an array includes a plurality of features. Forexample, an array includes between 4,000 and 10,000 features, or anyrange within 4,000 to 6000 features. For example, an array includesbetween 4,000 to 4,400 features, 4,000 to 4,800 features, 4,000 to 5,200features, 4,000 to 5,600 features, 5,600 to 6,000 features, 5,200 to6,000 features, 4,800 to 6,000 features, or 4,400 to 6,000 features. Insome embodiments, the array includes between 4,100 and 5,900 features,between 4,200 and 5,800 features, between 4,300 and 5,700 features,between 4,400 and 5,600 features, between 4,500 and 5,500 features,between 4,600 and 5,400 features, between 4,700 and 5,300 features,between 4,800 and 5,200 features, between 4,900 and 5,100 features, orany range within the disclosed sub-ranges. For example, the array caninclude about 4,000 features, about 4,200 features, about 4,400features, about 4,800 features, about 5,000 features, about 5,200features, about 5,400 features, about 5,600 features, or about 6,000features. In some embodiments, the array comprises at least 4,000features. In some embodiments, the array includes approximately 5,000features.

In some embodiments, features within an array have an irregulararrangement or relationship to one another, such that no discernablepattern or regularity is evident in the geometrical spacingrelationships among the features. For example, features within an arraymay be positioned randomly with respect to one another. Alternatively,features within an array may be positioned irregularly, but the spacingsmay be selected deterministically to ensure that the resultingarrangement of features is irregular.

In some embodiments, features within an array are positioned regularlywith respect to one another to form a pattern. A wide variety ofdifferent patterns of features can be implemented in arrays. Examples ofsuch patterns include, but are not limited to, square arrays offeatures, rectangular arrays of features, hexagonal arrays of features(including hexagonal close-packed arrays), radial arrays of features,spiral arrays of features, triangular arrays of features, and moregenerally, any array in which adjacent features in the array are reachedfrom one another by regular increments in linear and/or angularcoordinate dimensions.

In some embodiments, features within an array are positioned with adegree of regularity with respect to one another such that the array offeatures is neither perfectly regular nor perfectly irregular (i.e., thearray is “partially regular”). For example, in some embodiments,adjacent features in an array can be separated by a displacement in oneor more linear and/or angular coordinate dimensions that is 10% or more(e.g.., 20% or more, 30% or more, 40% or more, 50% or more, 60% or more,70% or more, 80% or more, 90% or more, 100% or more, 110% or more, 120%or more, 130% or more, 140% or more, 150% or more, 160% or more, 170% ormore, 180% or more, 190% or more, 200% or more) of an averagedisplacement or a nominal displacement between adjacent features in thearray. In certain embodiments, the distribution of displacements (linearand/or angular) between adjacent features in an array has a full-widthat half-maximum of between 0% and 200% (e.g., between 0% and 100%,between 0% and 75%, between 0% and 50%, between 0% and 25%, between 0%and 15%, between 0% and 10%) of an average displacement or nominaldisplacement between adjacent features in the array.

In some embodiments, arrays of features can have a variable geometry.For example, a first subset of features in an array can be arrangedaccording to a first geometrical pattern, and a second subset offeatures in the array can be arranged according to a second geometricalpattern that is different from the first pattern. Any of the patternsdescribed above can correspond to the first and/or second geometricalpatterns, for example.

In general, arrays of different feature densities can be prepared byadjusting the spacing between adjacent features in the array. In someembodiments, the geometric center-to-center spacing between adjacentfeatures in an array is between 100 nm and 100 µm. For example, thecenter-to-center spacing can be between 20 µm to 40 µm, 20 µm to 60 µm,20 µm to 80 µm, 80 µm to 100 µm, 60 µm to 100 µm, or 40 µm to 100 µm. Insome embodiments, the center-to-center spacing between adjacent arrayfeatures is between 30 µm and 100 µm, 40 µm and 90 µm, 50 µm and 80 µm,60 µm and 70 µm, 80 µm and 120 µm, or any range within the disclosedsub-ranges. In some embodiments, the center-to-center spacing betweenadjacent array features of a feature of an array is approximately 65 µm.

In some embodiments, an array of features can have a spatially varyingresolution. In general, an array with a spatially varying resolution isan array in which the center-to-center spacing (along linear, angular,or both linear and angular coordinate dimensions) between adjacentfeatures in the array varies. Such arrays can be useful in a variety ofapplications. For example, in some embodiments, depending upon thespatial resolution at which the sample is to be investigated, the samplecan be selectively associated with the portion of the array thatcorresponds approximately to the desired spatial resolution of themeasurement.

Arrays of spatially varying resolution can be implemented in a varietyof ways. In some embodiments, for example, the center-to-center spacingbetween adjacent features in the array varies continuously along one ormore linear and/or angular coordinate directions. Thus, for arectangular array, the spacing between successive rows of features,between successive columns of features, or between both successive rowsand successive columns of features, can vary continuously.

In certain embodiments, arrays of spatially varying resolution caninclude discrete domains with populations of features. Within eachdomain, adjacent features can have regular center-to-center spacings.Thus, for example, an array can include a first domain within whichadjacent features are spaced from one another along linear and/orangular coordinate dimensions by a first set of uniform coordinatedisplacements, and a second domain within which adjacent features arespaced from one another along linear and/or angular coordinatedimensions by a second set of uniform coordinate displacements. Thefirst and second sets of displacements differ in at least one coordinatedisplacement, such that adjacent features in the two domains are spaceddifferently, and the resolution of the array in the first domain istherefore different from the resolution of the array in the seconddomain.

In some embodiments, the center-to-center spacing of array features canbe sufficiently small such that array features are effectivelypositioned continuously or nearly continuously along one or more arraydimensions, with little or no displacement between array features alongthose dimensions. For example, in a feature array where the featurescorrespond to regions of a substrate (i.e., oligonucleotides aredirectly bound to the substrate), the displacement between adjacentoligonucleotides can be very small - effectively, the molecular width ofa single oligonucleotide. In such embodiments, each oligonucleotide caninclude a distinct spatial barcode such that the spatial location ofeach oligonucleotide in the array can be determined during sampleanalysis. Arrays of this type can have very high spatial resolution, butmay only include a single oligonucleotide corresponding to each distinctspatial location in a sample.

In general, the size of the array (which corresponds to the maximumdimension of the smallest boundary that encloses all features in thearray along one coordinate direction) can be selected as desired, basedon criteria such as the size of the sample, the feature sizes, and thedensity of capture probes within each feature. For example, in someembodiments, the array can be a rectangular or square array for whichthe maximum array dimension along each coordinate direction is 10 mm orless (e.g., 9 mm or less, 8 mm or less, 7 mm or less, 6 mm or less, 5 mmor less, 4 mm or less, 3 mm or less). Thus, for example, a square arrayof features can have dimensions of 8 mm by 8 mm, 7 mm by 7 mm, 5 mm by 5mm, or be smaller than 5 mm by 5 mm.

(E) Analyte Capture

In this section, general aspects of methods and systems for capturinganalytes are described. Individual method steps and system features canbe present in combination in many different embodiments; the specificcombinations described herein do not in any way limit other combinationsof steps and features.

Generally, analytes can be captured when contacting a biological samplewith, e.g., a substrate comprising capture probes (e.g., substrate withcapture probes embedded, spotted, printed on the substrate or asubstrate with features (e.g., beads, wells) comprising capture probes).

As used herein, “contact,” “contacted,” and/ or “contacting,” abiological sample with a substrate comprising features refers to anycontact (e.g., direct or indirect) such that capture probes can interact(e.g., capture) with analytes from the biological sample. For example,the substrate may be near or adjacent to the biological sample withoutdirect physical contact, yet capable of capturing analytes from thebiological sample. In some embodiments the biological sample is indirect physical contact with the substrate. In some embodiments, thebiological sample is in indirect physical contact with the substrate.For example, a liquid layer may be between the biological sample and thesubstrate. In some embodiments, the analytes diffuse through the liquidlayer. In some embodiments the capture probes diffuse through the liquidlayer. In some embodiments reagents may be delivered via the liquidlayer between the biological sample and the substrate. In someembodiments, indirect physical contact may be the presence of a secondsubstrate (e.g., a hydrogel, a film, a porous membrane) between thebiological sample and the first substrate comprising features withcapture probes. In some embodiments, reagents may be delivered by thesecond substrate to the biological sample.

Diffusion-Resistant Media / Lids

To increase efficiency by encouraging analyte diffusion toward thespatially-labelled capture probes, a diffusion-resistant medium can beused. In general, molecular diffusion of biological analytes occurs inall directions, including toward the capture probes (i.e. toward thespatially-barcoded array), and away from the capture probes (i.e. intothe bulk solution). Increasing diffusion toward the spatially-barcodedarray reduces analyte diffusion away from the spatially-barcoded arrayand increases the capturing efficiency of the capture probes.

In some embodiments, a biological sample is placed on the top of aspatially-barcoded substrate and a diffusion-resistant medium is placedon top of the biological sample. For example, the diffusion-resistantmedium can be placed onto an array that has been placed in contact witha biological sample. In some embodiments, the diffusion-resistant mediumand spatially-labelled array are the same component. For example, thediffusion-resistant medium can contain spatially-labelled capture probeswithin or on the diffusion-resistant medium (e.g., coverslip, slide,hydrogel, or membrane). In some embodiments, a sample is placed on asubstrate and a diffusion-resistant medium is placed on top of thebiological sample. Additionally, a spatially-barcoded capture probearray can be placed in close proximity over the diffusion-resistantmedium. For example, a diffusion-resistant medium may be sandwichedbetween a spatially-labelled array and a sample on a substrate. In someembodiments, the diffusion-resistant medium is disposed or spotted ontothe sample. In other embodiments, the diffusion-resistant medium isplaced in close proximity to the sample.

In general, the diffusion-resistant medium can be any material known tolimit diffusivity of biological analytes. For example, thediffusion-resistant medium can be a solid lid (e.g., coverslip or glassslide). In some embodiments, the diffusion-resistant medium may be madeof glass, silicon, paper, hydrogel polymer monoliths, or other material.In some embodiments, the glass side can be an acrylated glass slide. Insome embodiments, the diffusion-resistant medium is a porous membrane.In some embodiments, the material may be naturally porous. In someembodiments, the material may have pores or wells etched into solidmaterial. In some embodiments, the pore size can be manipulated tominimize loss of target analytes. In some embodiments, the membranechemistry can be manipulated to minimize loss of target analytes. Insome embodiments, the diffusion-resistant medium (i.e. hydrogel) iscovalently attached to a substrate (i.e. glass slide). In someembodiments, the diffusion-resistant medium can be any material known tolimit diffusivity of poly(A) transcripts. In some embodiments, thediffusion-resistant medium can be any material known to limit thediffusivity of proteins. In some embodiments, the diffusion-resistantmedium can be any material know to limit the diffusivity ofmacromolecular constituents.

In some embodiments, a diffusion-resistant medium includes one or morediffusion-resistant media. For example, one or more diffusion-resistantmedia can be combined in a variety of ways prior to placing the media incontact with a biological sample including, without limitation, coating,layering, or spotting. As another example, a hydrogel can be placed ontoa biological sample followed by placement of a lid (e.g., glass slide)on top of the hydrogel.

In some embodiments, a force (e.g., hydrodynamic pressure, ultrasonicvibration, solute contrasts, microwave radiation, vascular circulation,or other electrical, mechanical, magnetic, centrifugal, and/or thermalforces) is applied to control diffusion and enhance analyte capture. Insome embodiments, one or more forces and one or more diffusion-resistantmedia are used to control diffusion and enhance capture. For example, acentrifugal force and a glass slide can used contemporaneously. Any of avariety of combinations of a force and a diffusion-resistant medium canbe used to control or mitigate diffusion and enhance analyte capture.

In some embodiments, the diffusion-resistant medium, along with thespatially-barcoded array and sample, is submerged in a bulk solution. Insome embodiments, the bulk solution includes permeabilization reagents.In some embodiments, the diffusion-resistant medium includes at leastone permeabilization reagent. In some embodiments, thediffusion-resistant medium (i.e. hydrogel) is soaked in permeabilizationreagents before contacting the diffusion-resistant medium to the sample.In some embodiments, the diffusion-resistant medium can include wells(e.g., micro-, nano-, or picowells) containing a permeabilization bufferor reagents. In some embodiments, the diffusion-resistant medium caninclude permeabilization reagents. In some embodiments, thediffusion-resistant medium can contain dried reagents or monomers todeliver permeabilization reagents when the diffusion-resistant medium isapplied to a biological sample. In some embodiments, thediffusion-resistant medium is added to the spatially-barcoded array andsample assembly before the assembly is submerged in a bulk solution. Insome embodiments, the diffusion-resistant medium is added to thespatially-barcoded array and sample assembly after the sample has beenexposed to permeabilization reagents. In some embodiments, thepermeabilization reagents are flowed through a microfluidic chamber orchannel over the diffusion-resistant medium. In some embodiments, theflow controls the sample’s access to the permeabilization reagents. Insome embodiments, the target analytes diffuse out of the sample andtoward a bulk solution and get embedded in a spatially-labelled captureprobe-embedded diffusion-resistant medium. In some embodiments, a freesolution is sandwiched between the biological sample and adiffusion-resistant medium.

FIG. 13 is an illustration of an exemplary use of a diffusion-resistantmedium. A diffusion-resistant medium 1302 can be contacted with a sample1303. In FIG. 13 , a glass slide 1304 is populated withspatially-barcoded capture probes 1306, and the sample 1303, 1305 iscontacted with the array 1304, 1306. A diffusion-resistant medium 1302can be applied to the sample 1303, wherein the sample 1303 is sandwichedbetween a diffusion-resistant medium 1302 and a capture probe coatedslide 1304. When a permeabilization solution 1301 is applied to thesample, using the diffusion-resistant medium/lid 1302 directs migrationof the analytes 1305 toward the capture probes 1306 by reducingdiffusion of the analytes out into the medium. Alternatively, the lidmay contain permeabilization reagents.

Conditions for Capture

Capture probes on the substrate (or on a feature on the substrate)interact with released analytes through a capture domain, describedelsewhere, to capture analytes. In some embodiments, certain steps areperformed to enhance the transfer or capture of analytes by the captureprobes of the array. Examples of such modifications include, but are notlimited to, adjusting conditions for contacting the substrate with abiological sample (e.g., time, temperature, orientation, pH levels,pre-treating of biological samples, etc.), using force to transportanalytes (e.g., electrophoretic, centrifugal, mechanical, etc.),performing amplification reactions to increase the amount of biologicalanalytes (e.g., PCR amplification, in situ amplification, clonalamplification), and/or using labeled probes for detecting of ampliconsand barcodes.

In some embodiments, capture of analytes is facilitated by treating thebiological sample with permeabilization reagents. If a biological sampleis not permeabilized sufficiently, the amount of analyte captured on thesubstrate can be too low to enable adequate analysis. Conversely, if thebiological sample is too permeable, the analyte can diffuse away fromits origin in the biological sample, such that the relative spatialrelationship of the analytes within the biological sample is lost.Hence, a balance between permeabilizing the biological sample enough toobtain good signal intensity while still maintaining the spatialresolution of the analyte distribution in the biological sample isdesired. Methods of preparing biological samples to facilitation areknown in the art and can be modified depending on the biological sampleand how the biological sample is prepared (e.g., fresh frozen, FFPE,etc.).

Passive Capture Methods

In some embodiments, analytes can be migrated from a sample to asubstrate. Methods for facilitating migration can be passive (e.g.,diffusion) and/or active (e.g., electrophoretic migration of nucleicacids). Non-limiting examples of passive migration can include simplediffusion and osmotic pressure created by the rehydration of dehydratedobjects.

Passive migration by diffusion uses concentration gradients. Diffusionis movement of untethered objects toward equilibrium. Therefore, whenthere is a region of high object concentration and a region of lowobject concentration, the object (capture probe, the analyte, etc.)moves to an area of lower concentration. In some embodiments, untetheredanalytes move down a concentration gradient.

In some embodiments, different reagents may be added to the biologicalsample, such that the biological sample is rehydrated while improvingcapture of analytes. In some embodiments, the biological sample can berehydrated with permeabilization reagents. In some embodiments, thebiological sample can be rehydrated with a staining solution (e.g.,hematoxylin and eosin stain).

Active Capture Methods

In some examples of any of the methods described herein, an analyte in acell or a biological sample can be transported (e.g., passively oractively) to a capture probe (e.g., a capture probe affixed to a solidsurface).

For example, analytes in a cell or a biological sample can betransported to a capture probe (e.g., an immobilized capture probe)using an electric field (e.g., using electrophoresis), a pressuregradient, fluid flow, a chemical concentration gradient, a temperaturegradient, and/or a magnetic field. For example, analytes can betransported through, e.g., a gel (e.g., hydrogel matrix), a fluid, or apermeabilized cell, to a capture probe (e.g., an immobilized captureprobe).

In some examples, an electrophoretic field can be applied to analytes tofacilitate migration of the analytes towards a capture probe. In someexamples, a sample contacts a substrate and capture probes fixed on asubstrate (e.g., a slide, cover slip, or bead), and an electric currentis applied to promote the directional migration of charged analytestowards the capture probes fixed on the substrate. An electrophoresisassembly, where a cell or a biological sample is in contact with acathode and capture probes (e.g., capture probes fixed on a substrate),and where the capture probes (e.g., capture probes fixed on a substrate)is in contact with the cell or biological sample and an anode, can beused to apply the current.

Electrophoretic transfer of analytes can be performed while retainingthe relative spatial alignment of the analytes in the sample. As such,an analyte captured by the capture probes (e.g., capture probes fixed ona substrate) retains the spatial information of the cell or thebiological sample. Applying an electrophoretic field to analytes canalso result in an increase in temperature (e.g., heat). In someembodiments, the increased temperature (e.g., heat) can facilitate themigration of the analytes towards a capture probe.

In some examples, a spatially-addressable microelectrode array is usedfor spatially-constrained capture of at least one charged analyte ofinterest by a capture probe. The microelectrode array can be configuredto include a high density of discrete sites having a small area forapplying an electric field to promote the migration of chargedanalyte(s) of interest. For example, electrophoretic capture can beperformed on a region of interest using a spatially-addressablemicroelectrode array.

A high density of discrete sites on a microelectrode array can be usedfor small device. The surface can include any suitable density ofdiscrete sites (e.g., a density suitable for processing the sample onthe conductive substrate in a given amount of time). In an embodiment,the surface has a density of discrete sites greater than or equal toabout 500 sites per 1 mm². In some embodiments, the surface has adensity of discrete sites of about 100, about 200, about 300, about 400,about 500, about 600, about 700, about 800, about 900, about 1,000,about 2,000, about 3,000, about 4,000, about 5,000, about 6,000, about7,000, about 8,000, about 9,000, about 10,000, about 20,000, about40,000, about 60,000, about 80,000, about 100,000, or about 500,000sites per 1 mm². In some embodiments, the surface has a density ofdiscrete sites of at least about 200, at least about 300, at least about400, at least about 500, at least about 600, at least about 700, atleast about 800, at least about 900, at least about 1,000, at leastabout 2,000, at least about 3,000, at least about 4,000, at least about5,000, at least about 6,000, at least about 7,000, at least about 8,000,at least about 9,000, at least about 10,000, at least about 20,000, atleast about 40,000, at least about 60,000, at least about 80,000, atleast about 100,000, or at least about 500,000 sites per 1 mm².

Schematics illustrating an electrophoretic transfer system configured todirect transcript analytes toward a spatially-barcoded capture probearray are shown in FIG. 14A and FIG. 14B. In this exemplaryconfiguration of an electrophoretic system, a sample 1402 is sandwichedbetween the cathode 1401 and the spatially-barcoded capture probe array1404, 1405, and the spatially-barcoded capture probe array 1404, 1405 issandwiched between the sample 1402 and the anode 1403, such that thesample 1402, 1406 is in contact with the spatially-barcoded captureprobes 1407. When an electric field is applied to the electrophoretictransfer system, negatively charged mRNA analytes 1406 will be pulledtoward the positively charged anode 1403 and into the spatially-barcodedarray 1404, 1405 containing the spatially-barcoded capture probes 1407.The spatially-barcoded capture probes 1407 then interact with/hybridizewith/immobilize the mRNA target analytes 1406, making the analytecapture more efficient. The electrophoretic system set-up may changedepending on the target analyte. For example, proteins may be positive,negative, neutral, or polar depending on the protein as well as otherfactors (e.g. isoelectric point, solubility, etc.). The skilledpractitioner has the knowledge and experience to arrange theelectrophoretic transfer system to facilitate capture of a particulartarget analyte.

FIG. 15 is an illustration showing an exemplary workflow protocolutilizing an electrophoretic transfer system. In the example, Panel Adepicts a flexible spatially-barcoded feature array being contacted witha sample. The sample can be a flexible array, wherein the array isimmobilized on a hydrogel, membrane, or other flexible substrate. PanelB depicts contact of the array with the sample and imaging of thearray-sample assembly. The image of the sample/array assembly can beused to verify sample placement, choose a region of interest, or anyother reason for imaging a sample on an array as described herein. PanelC depicts application of an electric field using an electrophoretictransfer system to aid in efficient capture of a target analyte. Here,negatively charged mRNA target analytes migrate toward the positivelycharged anode. Panel D depicts application of reverse transcriptionreagents and first strand cDNA synthesis of the captured targetanalytes. Panel E depicts array removal and preparation for libraryconstruction (Panel F) and next-generation sequencing (Panel G).

Region of Interest

A biological sample can have regions that show morphological feature(s)that may indicate the presence of disease or the development of adisease phenotype. For example, morphological features at a specificsite within a tumor biopsy sample can indicate the aggressiveness,therapeutic resistance, metastatic potential, migration, stage,diagnosis, and/or prognosis of cancer in a subject. A change in themorphological features at a specific site within a tumor biopsy sampleoften correlate with a change in the level or expression of an analytein a cell within the specific site, which can, in turn, be used toprovide information regarding the aggressiveness, therapeuticresistance, metastatic potential, migration, stage, diagnosis, and/orprognosis of cancer in a subject. A region or area within a biologicalsample that is selected for specific analysis (e.g., a region in abiological sample that has morphological features of interest) is oftendescribed as “a region of interest.”

A region of interest in a biological sample can be used to analyze aspecific area of interest within a biological sample, and thereby, focusexperimentation and data gathering to a specific region of a biologicalsample (rather than an entire biological sample). This results inincreased time efficiency of the analysis of a biological sample.

A region of interest can be identified in a biological sample using avariety of different techniques, e.g., expansion microscopy, brightfield microscopy, dark field microscopy, phase contrast microscopy,electron microscopy, fluorescence microscopy, reflection microscopy,interference microscopy, confocal microscopy, and visual identification(e.g., by eye), and combinations thereof. For example, the staining andimaging of a biological sample can be performed to identify a region ofinterest. In some examples, the region of interest can correspond to aspecific structure of cytoarchitecture. In some embodiments, abiological sample can be stained prior to visualization to providecontrast between the different regions of the biological sample. Thetype of stain can be chosen depending on the type of biological sampleand the region of the cells to be stained. In some embodiments, morethan one stain can be used to visualize different aspects of thebiological sample, e.g., different regions of the sample, specific cellstructures (e.g. organelles), or different cell types. In otherembodiments, the biological sample can be visualized or imaged withoutstaining the biological sample.

In some embodiments, imaging can be performed using one or more fiducialmarkers, i.e., objects placed in the field of view of an imaging systemwhich appear in the image produced. Fiducial markers are typically usedas a point of reference or measurement scale. Fiducial markers caninclude, but are not limited to, detectable labels such as fluorescent,radioactive, chemiluminescent, and colorimetric labels. The use offiducial markers to stabilize and orient biological samples isdescribed, for example, in Carter et al., Applied Optics 46:421-427,2007), the entire contents of which are incorporated herein byreference. In some embodiments, a fiducial marker can be a physicalparticle (e.g., a nanoparticle, a microsphere, a nanosphere, a bead, orany of the other exemplary physical particles described herein or knownin the art).

In some embodiments, a fiducial marker can be present on a substrate toprovide orientation of the biological sample. In some embodiments, amicrosphere can be coupled to a substrate to aid in orientation of thebiological sample. In some examples, a microsphere coupled to asubstrate can produce an optical signal (e.g., fluorescence). In anotherexample, a microsphere can be attached to a portion (e.g., corner) of anarray in a specific pattern or design (e.g., hexagonal design) to aid inorientation of a biological sample on an array of features on thesubstrate. In some embodiments, a quantum dot can be coupled to thesubstrate to aid in the orientation of the biological sample. In someexamples, a quantum dot coupled to a substrate can produce an opticalsignal.

In some embodiments, a fiducial marker can be an immobilized moleculewith which a detectable signal molecule can interact to generate asignal. For example, a marker nucleic acid can be linked or coupled to achemical moiety capable of fluorescing when subjected to light of aspecific wavelength (or range of wavelengths). Such a marker nucleicacid molecule can be contacted with an array before, contemporaneouslywith, or after the tissue sample is stained to visualize or image thetissue section. Although not required, it can be advantageous to use amarker that can be detected using the same conditions (e.g., imagingconditions) used to detect a labelled cDNA.

In some embodiments, fiducial markers are included to facilitate theorientation of a tissue sample or an image thereof in relation to animmobilized capture probes on a substrate. Any number of methods formarking an array can be used such that a marker is detectable only whena tissue section is imaged. For instance, a molecule, e.g. a fluorescentmolecule that generates a signal, can be immobilized directly orindirectly on the surface of a substrate. Markers can be provided on asubstrate in a pattern (e.g., an edge, one or more rows, one or morelines, etc.).

In some embodiments, a fiducial marker can be randomly placed in thefield of view. For example, an oligonucleotide containing a fluorophorecan be randomly printed, stamped, synthesized, or attached to asubstrate (e.g., a glass slide) at a random position on the substrate. Atissue section can be contacted with the substrate such that theoligonucleotide containing the fluorophore contacts, or is in proximityto, a cell from the tissue section or a component of the cell (e.g., anmRNA or DNA molecule). An image of the substrate and the tissue sectioncan be obtained, and the position of the fluorophore within the tissuesection image can be determined (e.g., by reviewing an optical image ofthe tissue section overlaid with the fluorophore detection). In someembodiments, fiducial markers can be precisely placed in the field ofview (e.g., at known locations on a substrate). In this instance, afiducial marker can be stamped, attached, or synthesized on thesubstrate and contacted with a biological sample. Typically, an image ofthe sample and the fiducial marker is taken, and the position of thefiducial marker on the substrate can be confirmed by viewing the image.

In some embodiments, a fiducial marker can be an immobilized molecule(e.g., a physical particle) attached to the substrate. For example, afiducial marker can be a nanoparticle, e.g., a nanorod, a nanowire, ananocube, a nanopyramid, or a spherical nanoparticle. In some examples,the nanoparticle can be made of a heavy metal (e.g., gold). In someembodiments, the nanoparticle can be made from diamond. In someembodiments, the fiducial marker can be visible by eye.

As noted herein, any of the fiducial markers described herein (e.g.,microspheres, beads, or any of the other physical particles describedherein) can be located at a portion (e.g., corner) of an array in aspecific pattern or design (e.g., hexagonal design) to aid inorientation of a biological sample on an array of features on thesubstrate. In some embodiments, the fiducial markers located at aportion (e.g., corner) of an array (e.g., an array on a substrate) canbe pattern or designed in at least 1, at least 2, at least 3, or atleast 4 unique patterns. In some examples, the fiducial markers locatedat the corners of the array (e.g., an array on a substrate) can havefour unique patterns of fiducial markers.

In some examples, fiducial markers can surround the array. In someembodiments the fiducial markers allow for detection of, e.g.,mirroring. In some embodiments, the fiducial markers may completelysurround the array. In some embodiments, the fiducial markers may notcompletely surround the array. In some embodiments, the fiducial markersidentify the corners of the array. In some embodiments, one or morefiducial markers identify the center of the array. In some embodiments,the fiducial markers comprise patterned spots, wherein the diameter ofone or more patterned spot fiducial markers is approximately 100micrometers. The diameter of the fiducial markers can be any usefuldiameter including, but not limited to, 50 micrometers to 500micrometers in diameter. The fiducial markers may be arranged in such away that the center of one fiducial marker is between 100 micrometersand 200 micrometers from the center of one or more other fiducialmarkers surrounding the array. In some embodiments, the array with thesurrounding fiducial markers is approximately 8 mm by 8 mm. In someembodiments, the array without the surrounding fiducial markers issmaller than 8 mm by 50 mm.

In some embodiments, an array can be enclosed within a frame. Putanother way, the perimeter of an array can have fiducial markers suchthat the array is enclosed, or substantially enclosed. In someembodiments, the perimeter of an array can be fiducial markers (e.g.,any fiducial marker described herein). In some embodiments, theperimeter of an array can be uniform. For example, the fiducial markingscan connect, or substantially connect, consecutive corners of an arrayin such a fashion that the non-corner portion of the array perimeter isthe same on all sides (e.g., four sides) of the array. In someembodiments, the fiducial markers attached to the non-corner portions ofthe perimeter can be pattered or designed to aid in the orientation ofthe biological sample on the array. In some embodiments, the particlesattached to the non-corner portions of the perimeter can be patterned ordesigned in at least 1, at least 2, at least 3, or at least 4 patterns.In some embodiments, the patterns can have at least 2, at least 3, or atleast 4 unique patterns of fiducial markings on the non-corner portionof the array perimeter.

In some embodiments, an array can include at least two fiducial markers(e.g., at least 3, at least 4, at least 5, at least 6, at least 7, atleast 8, at least 9, at least 10, at least 12, at least 15, at least 20,at least 30, at least 40, at least 50, at least 60, at least 70, atleast 80, at least 90, at least 100 fiducial markers or more (e.g.,several hundred, several thousand, or tens of thousands of fiducialmarkers)) in distinct positions on the surface of a substrate. Fiducialmarkers can be provided on a substrate in a pattern (e.g., an edge, oneor more rows, one or more lines, etc.).

In some embodiments, staining and imaging a biological sample prior tocontacting the biological sample with a spatial array is performed toselect samples for spatial analysis. In some embodiments, the stainingincludes applying a fiducial marker as described above, includingfluorescent, radioactive, chemiluminescent, or colorimetric detectablemarkers. In some embodiments, the staining and imaging of biologicalsamples allows the user to identify the specific sample (or region ofinterest) the user wishes to assess.

In some embodiments, a lookup table (LUT) can be used to associate oneproperty with another property of a feature. These properties include,e.g., locations, barcodes (e.g., nucleic acid barcode molecules),spatial barcodes, optical labels, molecular tags, and other properties.

In some embodiments, a lookup table can associate a nucleic acid barcodemolecule with a feature. In some embodiments, an optical label of afeature can permit associating the feature with a biological particle(e.g., cell or nuclei). The association of a feature with a biologicalparticle can further permit associating a nucleic acid sequence of anucleic acid molecule of the biological particle to one or more physicalproperties of the biological particle (e.g., a type of a cell or alocation of the cell). For example, based on the relationship betweenthe barcode and the optical label, the optical label can be used todetermine the location of a feature, thus associating the location ofthe feature with the barcode sequence of the feature. Subsequentanalysis (e.g., sequencing) can associate the barcode sequence and theanalyte from the sample. Accordingly, based on the relationship betweenthe location and the barcode sequence, the location of the biologicalanalyte can be determined (e.g., in a specific type of cell or in a cellat a specific location of the biological sample).

In some embodiments, a feature can have a plurality of nucleic acidbarcode molecules attached thereto. The plurality of nucleic acidbarcode molecules can include barcode sequences. The plurality ofnucleic acid molecules attached to a given feature can have the samebarcode sequences, or two or more different barcode sequences. Differentbarcode sequences can be used to provide improved spatial locationaccuracy.

In some embodiments, a substrate is treated in order to minimize orreduce non-specific analyte hybridization within or between features.For example, treatment can include coating the substrate with ahydrogel, film, and/or membrane that creates a physical barrier tonon-specific hybridization. Any suitable hydrogel can be used. Forexample, hydrogel matrices prepared according to the methods set forthin U.S. Pat. Nos. 6,391,937, 9,512,422, and 9,889,422, and U.S. Pat.Application Publication Nos. U.S. 2017/0253918 and U.S. 2018/0052081,can be used. The entire contents of each of the foregoing documents areincorporated herein by reference.

Treatment can include adding a functional group that is reactive orcapable of being activated such that it becomes reactive after receivinga stimulus (e.g., photoreactive). Treatment can include treating withpolymers having one or more physical properties (e.g., mechanical,electrical, magnetic, and/or thermal) that minimize non-specific binding(e.g., that activate a substrate at certain locations to allow analytehybridization at those locations).

In some examples, an array (e.g., any of the exemplary arrays describedherein) can be contacted with only a portion of a biological sample(e.g., a cell, a feature, or a region of interest). In some examples, abiological sample is contacted with only a portion of an array (e.g.,any of the exemplary arrays described herein). In some examples, aportion of the array can be deactivated such that it does not interactwith the analytes in the biological sample (e.g., optical deactivation,chemical deactivation, heat deactivation, or blocking of the captureprobes in the array (e.g., using blocking probes)). In some examples, aregion of interest can be removed from a biological sample and then theregion of interest can be contacted to the array (e.g., any of thearrays described herein). A region of interest can be removed from abiological sample using microsurgery, laser capture microdissection,chunking, a microtome, dicing, trypsinization, labelling, and/orfluorescence-assisted cell sorting.

(F) Partitioning

As discussed above, in some embodiments, the sample can optionally beseparated into single cells, cell groups, or other fragments/pieces thatare smaller than the original, unfragmented sample. Each of thesesmaller portions of the sample can be analyzed to obtainspatially-resolved analyte information for the sample.

For samples that have been separated into smaller fragments - andparticularly, for samples that have been disaggregated, dissociated, orotherwise separated into individual cells -one method for analyzing thefragments involves partitioning the fragments into individual partitions(e.g., fluid droplets), and then analyzing the contents of thepartitions. In general, each partition maintains separation of its owncontents from the contents of other partitions. The partition can be adroplet in an emulsion, for example.

In addition to analytes, a partition can include additional components,and in particular, one or more beads. A partition can include a singlegel bead, a single cell bead, or both a single cell bead and single gelbead.

A partition can also include one or more reagents. Unique identifiers,such as barcodes, can be injected into the droplets previous to,subsequent to, or concurrently with droplet generation, such as via amicrocapsule (e.g., bead). Microfluidic channel networks (e.g., on achip) can be utilized to generate partitions. Alternative mechanisms canalso be employed in the partitioning of individual biological particles,including porous membranes through which aqueous mixtures of cells areextruded into non-aqueous fluids.

The partitions can be flowable within fluid streams. The partitions caninclude, for example, micro-vesicles that have an outer barriersurrounding an inner fluid center or core. In some cases, the partitionscan include a porous matrix that is capable of entraining and/orretaining materials within its matrix. The partitions can be droplets ofa first phase within a second phase, wherein the first and second phasesare immiscible. For example, the partitions can be droplets of aqueousfluid within a non-aqueous continuous phase (e.g., oil phase). Inanother example, the partitions can be droplets of a non-aqueous fluidwithin an aqueous phase. In some examples, the partitions can beprovided in a water-in-oil emulsion or oil-in-water emulsion. A varietyof different vessels are described in, for example, U.S. Pat.Application Publication No. 2014/0155295, the entire contents of whichare incorporated herein by reference. Emulsion systems for creatingstable droplets in non-aqueous or oil continuous phases are described,for example, in U.S. Pat. Application Publication No. 2010/0105112, theentire contents of which are incorporated herein by reference.

For droplets in an emulsion, allocating individual particles to discretepartitions can be accomplished, for example, by introducing a flowingstream of particles in an aqueous fluid into a flowing stream of anon-aqueous fluid, such that droplets are generated at the junction ofthe two streams. Fluid properties (e.g., fluid flow rates, fluidviscosities, etc.), particle properties (e.g., volume fraction, particlesize, particle concentration, etc.), microfluidic architectures (e.g.,channel geometry, etc.), and other parameters can be adjusted to controlthe occupancy of the resulting partitions (e.g., number of analytes perpartition, number of beads per partition, etc.). For example, partitionoccupancy can be controlled by providing the aqueous stream at a certainconcentration and/or flow rate of analytes.

To generate single analyte partitions, the relative flow rates of theimmiscible fluids can be selected such that, on average, the partitionscan contain less than one analyte per partition to ensure that thosepartitions that are occupied are primarily singly occupied. In somecases, partitions among a plurality of partitions can contain at mostone analyte. In some embodiments, the various parameters (e.g., fluidproperties, particle properties, microfluidic architectures, etc.) canbe selected or adjusted such that a majority of partitions are occupied,for example, allowing for only a small percentage of unoccupiedpartitions. The flows and channel architectures can be controlled as toensure a given number of singly occupied partitions, less than a certainlevel of unoccupied partitions and/or less than a certain level ofmultiply occupied partitions.

The channel segments described herein can be coupled to any of a varietyof different fluid sources or receiving components, includingreservoirs, tubing, manifolds, or fluidic components of other systems.As will be appreciated, the microfluidic channel structure can have avariety of geometries. For example, a microfluidic channel structure canhave one or more than one channel junction. As another example, amicrofluidic channel structure can have 2, 3, 4, or 5 channel segmentseach carrying particles that meet at a channel junction. Fluid can bedirected to flow along one or more channels or reservoirs via one ormore fluid flow units. A fluid flow unit can include compressors (e.g.,providing positive pressure), pumps (e.g., providing negative pressure),actuators, and the like to control flow of the fluid. Fluid can also orotherwise be controlled via applied pressure differentials, centrifugalforce, electrokinetic pumping, vacuum, capillary, and/or gravity flow.

A partition can include one or more unique identifiers, such asbarcodes. Barcodes can be previously, subsequently, or concurrentlydelivered to the partitions that hold the compartmentalized orpartitioned biological particle. For example, barcodes can be injectedinto droplets previous to, subsequent to, or concurrently with dropletgeneration. The delivery of the barcodes to a particular partitionallows for the later attribution of the characteristics of theindividual biological particle to the particular partition. Barcodes canbe delivered, for example on a nucleic acid molecule (e.g., anoligonucleotide), to a partition via any suitable mechanism. Barcodednucleic acid molecules can be delivered to a partition via amicrocapsule. A microcapsule, in some instances, can include a bead.

In some embodiments, barcoded nucleic acid molecules can be initiallyassociated with the microcapsule and then released from themicrocapsule. Release of the barcoded nucleic acid molecules can bepassive (e.g., by diffusion out of the microcapsule). In addition oralternatively, release from the microcapsule can be upon application ofa stimulus which allows the barcoded nucleic acid nucleic acid moleculesto dissociate or to be released from the microcapsule. Such stimulus candisrupt the microcapsule, an interaction that couples the barcodednucleic acid molecules to or within the microcapsule, or both. Suchstimulus can include, for example, a thermal stimulus, photo-stimulus,chemical stimulus (e.g., change in pH or use of a reducing agent(s)), amechanical stimulus, a radiation stimulus; a biological stimulus (e.g.,enzyme), or any combination thereof.

In some embodiments, one more barcodes (e.g., spatial barcodes, UMIs, ora combination thereof) can be introduced into a partition as part of theanalyte. As described previously, barcodes can be bound to the analytedirectly, or can form part of a capture probe or analyte capture agentthat is hybridized to, conjugated to, or otherwise associated with ananalyte, such that when the analyte is introduced into the partition,the barcode(s) are introduced as well. As described above, FIG. 16 showsan example of a microfluidical channel structure for partitioningindividual analytes (e.g., cells) into discrete partitions.

FIG. 16 shows an example of a microfluidic channel structure forpartitioning individual analytes (e.g., cells) into discrete partitions.The channel structure can include channel segments 1601, 1602, 1603, and1604 communicating at a channel junction 1605. In operation, a firstaqueous fluid 1606 that includes suspended biological particles (orcells) 1607 may be transported along channel segment 1601 into junction1605, while a second fluid 1608 that is immiscible with the aqueousfluid 1606 is delivered to the junction 1605 from each of channelsegments 1602 and 1603 to create discrete droplets 1609, 1610 of thefirst aqueous fluid 1606 flowing into channel segment 1604, and flowingaway from junction 1605. The channel segment 1604 may be fluidicallycoupled to an outlet reservoir where the discrete droplets can be storedand/or harvested. A discrete droplet generated may include an individualbiological particle 1607 (such as droplets 1609). A discrete dropletgenerated may include more than one individual biological particle 1607.A discrete droplet may contain no biological particle 1607 (such asdroplet 1610). Each discrete partition may maintain separation of itsown contents (e.g., individual biological particle 1607) from thecontents of other partitions.

FIG. 17A shows another example of a microfluidic channel structure 1700for delivering beads to droplets. The channel structure includes channelsegments 1701, 1702, 1703, 1704 and 1705 communicating at a channeljunction 1706. During operation, the channel segment 1701 can transportan aqueous fluid 1707 that includes a plurality of beads 1708 along thechannel segment 1701 into junction 1706. The plurality of beads 1708 canbe sourced from a suspension of beads. For example, the channel segment1701 can be connected to a reservoir that includes an aqueous suspensionof beads 1708. The channel segment 1702 can transport the aqueous fluid1707 that includes a plurality of particles 1709 (e.g., cells) along thechannel segment 1702 into junction 1706. In some embodiments, theaqueous fluid 1707 in either the first channel segment 1701 or thesecond channel segment 1702, or in both segments, can include one ormore reagents, as further described below.

A second fluid 1710 that is immiscible with the aqueous fluid 1707(e.g., oil) can be delivered to the junction 1706 from each of channelsegments 1703 and 1704. Upon meeting of the aqueous fluid 1707 from eachof channel segments 1701 and 1702 and the second fluid 1710 from each ofchannel segments 1703 and 1704 at the channel junction 1706, the aqueousfluid 1707 can be partitioned as discrete droplets 1711 in the secondfluid 1710 and flow away from the junction 1706 along channel segment1705. The channel segment 1705 can deliver the discrete droplets to anoutlet reservoir fluidly coupled to the channel segment 1705, where theycan be harvested.

As an alternative, the channel segments 1701 and 1702 can meet atanother junction upstream of the junction 1706. At such junction, beadsand biological particles can form a mixture that is directed alonganother channel to the junction 1706 to yield droplets 1711. The mixturecan provide the beads and biological particles in an alternatingfashion, such that, for example, a droplet includes a single bead and asingle biological particle.

The second fluid 1710 can include an oil, such as a fluorinated oil,that includes a fluorosurfactant for stabilizing the resulting droplets,for example, inhibiting subsequent coalescence of the resulting droplets1711.

The partitions described herein can include small volumes, for example,less than about 10 microliters (TL), 5 TL, 1 TL, 900 picoliters (pL),800 pL, 700 pL, 600 pL, 500 pL, 400 pL, 300 pL, 200 pL, 100 pL, 50 pL,20 pL, 10 pL, 1 pL, 500 nanoliters (nL), 100 nL, 50 nL, or less. In theforegoing discussion, droplets with beads were formed at the junction ofdifferent fluid streams. In some embodiments, droplets can be formed bygravity-based partitioning methods.

FIG. 17B shows a cross-section view of another example of a microfluidicchannel structure 1750 with a geometric feature for controlledpartitioning. A channel structure 1750 can include a channel segment1752 communicating at a channel junction 1758 (or intersection) with areservoir 1754. In some instances, the channel structure 1750 and one ormore of its components can correspond to the channel structure 1700 andone or more of its components.

An aqueous fluid 1760 comprising a plurality of particles 1756 may betransported along the channel segment 1752 into the junction 1758 tomeet a second fluid 1762 (e.g., oil, etc.) that is immiscible with theaqueous fluid 1760 in the reservoir 1754 to create droplets 1764 of theaqueous fluid 1760 flowing into the reservoir 1754. At the junction 1758where the aqueous fluid 1760 and the second fluid 1762 meet, dropletscan form based on factors such as the hydrodynamic forces at thejunction 1758, relative flow rates of the two fluids 1760, 1762, fluidproperties, and certain geometric parameters (e.g., Δh, etc.) of thechannel structure 1750. A plurality of droplets can be collected in thereservoir 1754 by continuously injecting the aqueous fluid 1760 from thechannel segment 1752 at the junction 1758.

A discrete droplet generated may comprise one or more particles of theplurality of particles 1756. As described elsewhere herein, a particlemay be any particle, such as a bead, cell bead, gel bead, biologicalparticle, macromolecular constituents of biological particle, or otherparticles. Alternatively, a discrete droplet generated may not includeany particles.

In some instances, the aqueous fluid 1760 can have a substantiallyuniform concentration or frequency of particles 1756. As describedelsewhere herein, the particles 1756 (e.g., beads) can be introducedinto the channel segment 1752 from a separate channel (not shown in FIG.17 ). The frequency of particles 1756 in the channel segment 1752 may becontrolled by controlling the frequency in which the particles 1756 areintroduced into the channel segment 1752 and/or the relative flow ratesof the fluids in the channel segment 1752 and the separate channel. Insome instances, the particles 1756 can be introduced into the channelsegment 1752 from a plurality of different channels, and the frequencycontrolled accordingly. In some instances, different particles may beintroduced via separate channels. For example, a first separate channelcan introduce beads and a second separate channel can introducebiological particles into the channel segment 1752. The first separatechannel introducing the beads may be upstream or downstream of thesecond separate channel introducing the biological particles.

In some instances, the second fluid 1762 may not be subjected to and/ordirected to any flow in or out of the reservoir 1754. For example, thesecond fluid 1762 may be substantially stationary in the reservoir 1754.In some instances, the second fluid 1762 may be subjected to flow withinthe reservoir 1754, but not in or out of the reservoir 1754, such as viaapplication of pressure to the reservoir 1754 and/or as affected by theincoming flow of the aqueous fluid 1760 at the junction 1758.Alternatively, the second fluid 1762 may be subjected and/or directed toflow in or out of the reservoir 1754. For example, the reservoir 1754can be a channel directing the second fluid 1762 from upstream todownstream, transporting the generated droplets.

The channel structure 1750 at or near the junction 1758 may have certaingeometric features that at least partly determine the sizes and/orshapes of the droplets formed by the channel structure 1750. The channelsegment 1752 can have a first cross-section height, h1, and thereservoir 1754 can have a second cross-section height, h2. The firstcross-section height, h1, and the second cross-section height, h2, maybe different, such that at the junction 1758, there is a heightdifference of Δh. The second cross-section height, h2, may be greaterthan the first cross-section height, h1. In some instances, thereservoir may thereafter gradually increase in cross-section height, forexample, the more distant it is from the junction 1758. In someinstances, the cross-section height of the reservoir may increase inaccordance with expansion angle, β, at or near the junction 1758. Theheight difference, Δh, and/or expansion angle, β, can allow the tongue(portion of the aqueous fluid 1760 leaving channel segment 1752 atjunction 1758 and entering the reservoir 1754 before droplet formation)to increase in depth and facilitate decrease in curvature of theintermediately formed droplet. For example, droplet size may decreasewith increasing height difference and/or increasing expansion angle.

The height difference, Δh, can be at least about 1 µm. Alternatively,the height difference can be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60,70, 80, 90, 100, 200, 300, 400, 500 µm or more. Alternatively, theheight difference can be at most about 500, 400, 300, 200, 100, 90, 80,70, 60, 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11,10, 9, 8, 7, 6, 5, 4, 3, 2, 1 µm or less. In some instances, theexpansion angle, β, may be between a range of from about 0.5° to about4°, from about 0.1° to about 10°, or from about 0° to about 90°. Forexample, the expansion angle can be at least about 0.01°, 0.1°, 0.2°,0.3°, 0.4°, 0.5°, 0.6°, 0.7°, 0.8°, 0.9°, 1°, 2°, 3°, 4°, 5°, 6°, 7°,8°, 9°, 10°, 15°, 20°, 25°, 30°, 35°, 40°, 45°, 50°, 55°, 60°, 65°, 70°,75°, 80°, 85°, or higher. In some instances, the expansion angle can beat most about 89°, 88°, 87°, 86°, 85°, 84°, 83°, 82°, 81°, 80°, 75°,70°, 65°, 60°, 55°, 50°, 45°, 40°, 35°, 30°, 25°, 20°, 15°, 10°, 9°, 8°,7°, 6°, 5°, 4°, 3°, 2°, 1°, 0.1°, 0.01°, or less.

In some instances, the flow rate of the aqueous fluid 1760 entering thejunction 1758 can be between about 0.04 microliters (µL)/minute (min)and about 40 µL/min. In some instances, the flow rate of the aqueousfluid 1760 entering the junction 1758 can be between about 0.01microliters (µL)/minute (min) and about 100 µL/min. Alternatively, theflow rate of the aqueous fluid 1760 entering the junction 1758 can beless than about 0.01 µL/min. alternatively, the flow rate of the aqueousfluid 1760 entering the junction 1758 can be greater than about 40µL/min, such as 45 µL/min, 50 µL/min, 55 µL/min, 60 µL/min, 65 µL/min,70 µL/min, 75 µL/min,80 µL/min, 85 µL/min, 90 µL/min, 95 µL/min, 100µL/min, 110 µL/min, 120 µL/min, 130 µL/min, 140 µL/min, 150 µL/min, orgreater. At lower flow rates, such as flow rates of about less than orequal to 10 microliters/minute, the droplet radius may not be dependenton the flow rate of the aqueous fluid 1760 entering the junction 1758.The second fluid 1762 may be stationary, or substantially stationary, inthe reservoir 1754. Alternatively, the second fluid 1762 may be flowing,such as at the above flow rates described for the aqueous fluid 1760.

While FIG. 17B illustrates the height difference, Δh, being abrupt atthe junction 1758 (e.g., a step increase), the height difference mayincrease gradually (e.g., from about 0 µm to a maximum heightdifference). Alternatively, the height difference may decrease gradually(e.g., taper) from a maximum height difference. A gradual increase ordecrease in height difference, as used herein, may refer to a continuousincremental increase or decrease in height difference, wherein an anglebetween any one differential segment of a height profile and animmediately adjacent differential segment of the height profile isgreater than 90°. For example, at the junction 1758, a bottom wall ofthe channel and a bottom wall of the reservoir can meet at an anglegreater than 90°. Alternatively or in addition, a top wall (e.g.,ceiling) of the channel and a top wall (e.g., ceiling) of the reservoircan meet an angle greater than 90°. A gradual increase or decrease maybe linear or non-linear (e.g., exponential, sinusoidal, etc.).Alternatively or in addition, the height difference may variablyincrease and/or decrease linearly or non-linearly. While FIG. 17Billustrates the expanding reservoir cross-section height as linear(e.g., constant expansion angle, β), the cross-section height may expandnon-linearly. For example, the reservoir may be defined at leastpartially by a dome-like (e.g., hemispherical) shape having variableexpansion angles. The cross-section height may expand in any shape.

A variety of different beads can be incorporated into partitions asdescribed above. In some embodiments, for example, non-barcoded beadscan be incorporated into the partitions. For example, where thebiological particle (e.g., a cell) that is incorporated into thepartitions carries one or more barcodes (e.g., spatial barcode(s),UMI(s), and combinations thereof), the bead can be a non-barcoded bead.

In some embodiments, a barcode carrying bead can be incorporated intopartitions. For example, a nucleic acid molecule, such as anoligonucleotide, can be coupled to a bead by a releasable linkage, suchas, for example, a disulfide linker. The same bead can be coupled (e.g.,via releasable linkage) to one or more other nucleic acid molecules. Thenucleic acid molecule can be or include a barcode. As noted elsewhereherein, the structure of the barcode can include a number of sequenceelements.

The nucleic acid molecule can include a functional domain that can beused in subsequent processing. For example, the functional domain caninclude one or more of a sequencer specific flow cell attachmentsequence (e.g., a P5 sequence for Illumina® sequencing systems) and asequencing primer sequence (e.g., a R1 primer for Illumina® sequencingsystems). The nucleic acid molecule can include a barcode sequence foruse in barcoding the sample (e.g., DNA, RNA, protein, etc.). In somecases, the barcode sequence can be bead-specific such that the barcodesequence is common to all nucleic acid molecules coupled to the samebead. Alternatively or in addition, the barcode sequence can bepartition-specific such that the barcode sequence is common to allnucleic acid molecules coupled to one or more beads that are partitionedinto the same partition. The nucleic acid molecule can include aspecific priming sequence, such as an mRNA specific priming sequence(e.g., poly(T) sequence), a targeted priming sequence, and/or a randompriming sequence. The nucleic acid molecule can include an anchoringsequence to ensure that the specific priming sequence hybridizes at thesequence end (e.g., of the mRNA). For example, the anchoring sequencecan include a random short sequence of nucleotides, such as a 1-mer,2-mer, 3-mer or longer sequence, which can ensure that a poly(T) segmentis more likely to hybridize at the sequence end of the poly(A) tail ofthe mRNA.

The nucleic acid molecule can include a unique molecular identifyingsequence (e.g., unique molecular identifier (UMI)). In some embodiments,the unique molecular identifying sequence can include from about 5 toabout 8 nucleotides. Alternatively, the unique molecular identifyingsequence can include less than about 5 or more than about 8 nucleotides.The unique molecular identifying sequence can be a unique sequence thatvaries across individual nucleic acid molecules coupled to a singlebead.

In some embodiments, the unique molecular identifying sequence can be arandom sequence (e.g., such as a random N-mer sequence). For example,the UMI can provide a unique identifier of the starting mRNA moleculethat was captured, in order to allow quantitation of the number oforiginal expressed RNA.

In general, an individual bead can be coupled to any number ofindividual nucleic acid molecules, for example, from one to tens tohundreds of thousands or even millions of individual nucleic acidmolecules. The respective barcodes for the individual nucleic acidmolecules can include both common sequence segments or relatively commonsequence segments and variable or unique sequence segments betweendifferent individual nucleic acid molecules coupled to the same bead.

FIG. 17C depicts a workflow wherein cells are partitioned into dropletsalong with barcode-bearing beads 1770. See FIG. 17A. The droplet formsan isolated reaction chamber wherein the cells can be lysed 1771 andtarget analytes within the cells can then be captured 1772 and amplified1773, 1774 according to previously described methods. After sequencelibrary preparation clean-up 1775, the material is sequenced and/orquantified 1776 according to methods described herein.

It should be noted that while the example workflow in FIG. 17C includessteps specifically for the analysis of mRNA, analogous workflows can beimplemented for a wide variety of other analytes, including any of theanalytes described previously.

By way of example, in the context of analyzing sample RNA as shown inFIG. 17C, the poly(T) segment of one of the released nucleic acidmolecules (e.g., from the bead) can hybridize to the poly(A) tail of amRNA molecule. Reverse transcription can result in a cDNA transcript ofthe mRNA, which transcript includes each of the sequence segments of thenucleic acid molecule. If the nucleic acid molecule includes ananchoring sequence, it will more likely hybridize to and prime reversetranscription at the sequence end of the poly(A) tail of the mRNA.

Within any given partition, all of the cDNA transcripts of theindividual mRNA molecules can include a common barcode sequence segment.However, the transcripts made from the different mRNA molecules within agiven partition can vary at the unique molecular identifying sequencesegment (e.g., UMI segment). Beneficially, even following any subsequentamplification of the contents of a given partition, the number ofdifferent UMIs can be indicative of the quantity of mRNA originatingfrom a given partition. As noted above, the transcripts can beamplified, cleaned up and sequenced to identify the sequence of the cDNAtranscript of the mRNA, as well as to sequence the barcode segment andthe UMI segment. While a poly(T) primer sequence is described, othertargeted or random priming sequences can also be used in priming thereverse transcription reaction. Likewise, although described asreleasing the barcoded oligonucleotides into the partition, in somecases, the nucleic acid molecules bound to the bead can be used tohybridize and capture the mRNA on the solid phase of the bead, forexample, in order to facilitate the separation of the RNA from othercell contents.

In some embodiments, precursors that include a functional group that isreactive or capable of being activated such that it becomes reactive canbe polymerized with other precursors to generate gel beads that includethe activated or activatable functional group. The functional group canthen be used to attach additional species (e.g., disulfide linkers,primers, other oligonucleotides, etc.) to the gel beads. For example,some precursors featuring a carboxylic acid (COOH) group canco-polymerize with other precursors to form a bead that also includes aCOOH functional group. In some cases, acrylic acid (a species comprisingfree COOH groups), acrylamide, and bis(acryloyl)cystamine can beco-polymerized together to generate a bead with free COOH groups. TheCOOH groups of the bead can be activated (e.g., via1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) andN-Hydroxysuccinimide (NHS) or4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride(DMTMM)) such that they are reactive (e.g., reactive to amine functionalgroups where EDC/NHS or DMTMM are used for activation). The activatedCOOH groups can then react with an appropriate species (e.g., a speciescomprising an amine functional group where the carboxylic acid groupsare activated to be reactive with an amine functional group) comprisinga moiety to be linked to the bead.

In some embodiments, a degradable bead can be introduced into apartition, such that the bead degrades within the partition and anyassociated species (e.g., oligonucleotides) are released within thedroplet when the appropriate stimulus is applied. The free species(e.g., oligonucleotides, nucleic acid molecules) can interact with otherreagents contained in the partition. For example, a polyacrylamide beadfeaturing cystamine and linked, via a disulfide bond, to a barcodesequence, can be combined with a reducing agent within a droplet of awater-in-oil emulsion. Within the droplet, the reducing agent can breakthe various disulfide bonds, resulting in bead degradation and releaseof the barcode sequence into the aqueous, inner environment of thedroplet. In another example, heating of a droplet with a bead-boundbarcode sequence in basic solution can also result in bead degradationand release of the attached barcode sequence into the aqueous, innerenvironment of the droplet.

Any suitable number of species (e.g., primer, barcoded oligonucleotide)can be associated with a bead such that, upon release from the bead, thespecies (e.g., primer, e.g., barcoded oligonucleotide) are present inthe partition at a pre-defined concentration. Such pre-definedconcentration can be selected to facilitate certain reactions forgenerating a sequencing library, e.g., amplification, within thepartition. In some cases, the pre-defined concentration of the primercan be limited by the process of producing nucleic acid molecule (e.g.,oligonucleotide) bearing beads.

A degradable bead can include one or more species with a labile bondsuch that, when the bead/species is exposed to the appropriate stimulus,the bond is broken and the bead degrades. The labile bond can be achemical bond (e.g., covalent bond, ionic bond) or can be another typeof physical interaction (e.g., van der Waals interactions, dipole-dipoleinteractions, etc.) In some embodiments, a crosslinker used to generatea bead can include a labile bond. Upon exposure to the appropriateconditions, the labile bond can be broken and the bead degraded. Forexample, upon exposure of a polyacrylamide gel bead that includescystamine crosslinkers to a reducing agent, the disulfide bonds of thecystamine can be broken and the bead degraded. A degradable bead can beuseful in more quickly releasing an attached species (e.g., a nucleicacid molecule, a barcode sequence, a primer, etc.) from the bead whenthe appropriate stimulus is applied to the bead as compared to a beadthat does not degrade. For example, for a species bound to an innersurface of a porous bead or in the case of an encapsulated species, thespecies can have greater mobility and accessibility to other species insolution upon degradation of the bead. In some embodiments, a speciescan also be attached to a degradable bead via a degradable linker (e.g.,disulfide linker). The degradable linker can respond to the same stimulias the degradable bead or the two degradable species can respond todifferent stimuli. For example, a barcode sequence can be attached, viaa disulfide bond, to a polyacrylamide bead comprising cystamine. Uponexposure of the barcoded-bead to a reducing agent, the bead degrades andthe barcode sequence is released upon breakage of both the disulfidelinkage between the barcode sequence and the bead and the disulfidelinkages of the cystamine in the bead.

As will be appreciated from the above description, while referred to asdegradation of a bead, in many embodiments, degradation can refer to thedisassociation of a bound or entrained species from a bead, both withand without structurally degrading the physical bead itself. Forexample, entrained species can be released from beads through osmoticpressure differences due to, for example, changing chemicalenvironments. By way of example, alteration of bead pore sizes due toosmotic pressure differences can generally occur without structuraldegradation of the bead itself. In some cases, an increase in pore sizedue to osmotic swelling of a bead can permit the release of entrainedspecies within the bead. In some embodiments, osmotic shrinking of abead can cause a bead to better retain an entrained species due to poresize contraction. Numerous chemical triggers can be used to trigger thedegradation of beads within partitions. Examples of these chemicalchanges can include, but are not limited to pH-mediated changes to theintegrity of a component within the bead, degradation of a component ofa bead via cleavage of cross-linked bonds, and depolymerization of acomponent of a bead.

In some embodiments, a bead can be formed from materials that includedegradable chemical cross-linkers, such as BAC or cystamine. Degradationof such degradable cross-linkers can be accomplished through a number ofmechanisms. In some examples, a bead can be contacted with a chemicaldegrading agent that can induce oxidation, reduction or other chemicalchanges. For example, a chemical degrading agent can be a reducingagent, such as dithiothreitol (DTT). Additional examples of reducingagents can include β-mercaptoethanol, (2S)-2-amino-1,4-dimercaptobutane(dithiobutylamine or DTBA), tris(2-carboxyethyl) phosphine (TCEP), orcombinations thereof. A reducing agent can degrade the disulfide bondsformed between gel precursors forming the bead, and thus, degrade thebead.

In certain embodiments, a change in pH of a solution, such as anincrease in pH, can trigger degradation of a bead. In other embodiments,exposure to an aqueous solution, such as water, can trigger hydrolyticdegradation, and thus degradation of the bead. In some cases, anycombination of stimuli can trigger degradation of a bead. For example, achange in pH can enable a chemical agent (e.g., DTT) to become aneffective reducing agent.

Beads can also be induced to release their contents upon the applicationof a thermal stimulus. A change in temperature can cause a variety ofchanges to a bead. For example, heat can cause a solid bead to liquefy.A change in heat can cause melting of a bead such that a portion of thebead degrades. In other cases, heat can increase the internal pressureof the bead components such that the bead ruptures or explodes. Heat canalso act upon heat-sensitive polymers used as materials to constructbeads.

In addition to beads and analytes, partitions that are formed caninclude a variety of different reagents and species. For example, whenlysis reagents are present within the partitions, the lysis reagents canfacilitate the release of analytes within the partition. Examples oflysis agents include bioactive reagents, such as lysis enzymes that areused for lysis of different cell types, e.g., gram positive or negativebacteria, plants, yeast, mammalian, etc., such as lysozymes,achromopeptidase, lysostaphin, labiase, kitalase, lyticase, and avariety of other lysis enzymes available from, e.g., Sigma-Aldrich, Inc.(St. Louis, MO), as well as other commercially available lysis enzymes.Other lysis agents can additionally or alternatively be co-partitionedto cause the release analytes into the partitions. For example, in somecases, surfactant-based lysis solutions can be used to lyse cells,although these can be less desirable for emulsion based systems wherethe surfactants can interfere with stable emulsions. In someembodiments, lysis solutions can include non-ionic surfactants such as,for example, TritonX-100 and Tween 20. In some embodiments, lysissolutions can include ionic surfactants such as, for example, sarcosyland sodium dodecyl sulfate (SDS). Electroporation, thermal, acoustic ormechanical cellular disruption can also be used in certain embodiments,e.g., non-emulsion based partitioning such as encapsulation of analytesthat can be in addition to or in place of droplet partitioning, whereany pore size of the encapsulate is sufficiently small to retain nucleicacid fragments of a given size, following cellular disruption.

Examples of other species that can be co-partitioned with analytes inthe partitions include, but are not limited to, DNase and RNaseinactivating agents or inhibitors, such as proteinase K, chelatingagents, such as EDTA, and other reagents employed in removing orotherwise reducing negative activity or impact of different cell lysatecomponents on subsequent processing of nucleic acids. Additionalreagents can also be co-partitioned, including endonucleases to fragmentDNA, DNA polymerase enzymes and dNTPs used to amplify nucleic acidfragments and to attach the barcode molecular tags to the amplifiedfragments.

Additional reagents can also include reverse transcriptase enzymes,including enzymes with terminal transferase activity, primers andoligonucleotides, and switch oligonucleotides (also referred to hereinas “switch oligos” or “template switching oligonucleotides”) which canbe used for template switching. In some embodiments, template switchingcan be used to increase the length of a cDNA. Template switching can beused to append a predefined nucleic acid sequence to the cDNA. In anexample of template switching, cDNA can be generated from reversetranscription of a template, e.g., cellular mRNA, where a reversetranscriptase with terminal transferase activity can add additionalnucleotides, e.g., poly(C), to the cDNA in a template independentmanner. Switch oligos can include sequences complementary to theadditional nucleotides, e.g., poly(G). The additional nucleotides (e.g.,poly(C)) on the cDNA can hybridize to the additional nucleotides (e.g.,poly(G)) on the switch oligo, whereby the switch oligo can be used bythe reverse transcriptase as template to further extend the cDNA.Template switching oligonucleotides can include a hybridization regionand a template region. The hybridization region can include any sequencecapable of hybridizing to the target. In some cases, the hybridizationregion includes a series of G bases to complement the overhanging Cbases at the 3′ end of a cDNA molecule. The series of G bases caninclude 1 G base, 2 G bases, 3 G bases, 4 G bases, 5 G bases or morethan 5 G bases. The template sequence can include any sequence to beincorporated into the cDNA. In some cases, the template region includesat least 1 (e.g., at least 2, 3, 4, 5 or more) tag sequences and/orfunctional sequences. Switch oligos can include deoxyribonucleic acids;ribonucleic acids; bridged nucleic acids, modified nucleic acidsincluding 2-Aminopurine, 2,6-Diaminopurine (2-Amino-dA), inverted dT,5-Methyl dC, 2′-deoxyInosine, Super T (5-hydroxybutynl-2′-deoxyuridine),Super G (8-aza-7-deazaguanosine), locked nucleic acids (LNAs), unlockednucleic acids (UNAs, e.g., UNA-A, UNA-U, UNA-C, UNA-G), Iso-dG, Iso-dC,2′ Fluoro bases (e.g., Fluoro C, Fluoro U, Fluoro A, and Fluoro G), andcombinations of the foregoing.

In some embodiments, beads that are partitioned with the analyte caninclude different types of oligonucleotides bound to the bead, where thedifferent types of oligonucleotides bind to different types of analytes.For example, a bead can include one or more first oligonucleotides(which can be capture probes, for example) that can bind or hybridize toa first type of analyte, such as mRNA for example, and one or moresecond oligonucleotides (which can be capture probes, for example) thatcan bind or hybridize to a second type of analyte, such as gDNA forexample. Partitions can also include lysis agents that aid in releasingnucleic acids from the co-partitioned cell, and can also include anagent (e.g., a reducing agent) that can degrade the bead and/or breakcovalent linkages between the oligonucleotides and the bead, releasingthe oligonucleotides into the partition. The released barcodedoligonucleotides (which can also be barcoded) can hybridize with mRNAreleased from the cell and also with gDNA released from the cell.

Barcoded constructs thus formed from hybridization can include a firsttype of construct that includes a sequence corresponding to an originalbarcode sequence from the bead and a sequence corresponding to atranscript from the cell, and a second type of construct that includes asequence corresponding to the original barcode sequence from the beadand a sequence corresponding to genomic DNA from the cell. The barcodedconstructs can then be released/removed from the partition and, in someembodiments, further processed to add any additional sequences. Theresulting constructs can then be sequenced, the sequencing dataprocessed, and the results used to spatially characterize the mRNA andthe gDNA from the cell.

In another example, a partition includes a bead that includes a firsttype of oligonucleotide (e.g., a first capture probe) with a firstbarcode sequence, a poly(T) priming sequence that can hybridize with thepoly(A) tail of an mRNA transcript, and a UMI barcode sequence that canuniquely identify a given transcript. The bead also includes a secondtype of oligonucleotide (e.g., a second capture probe) with a secondbarcode sequence, a targeted priming sequence that is capable ofspecifically hybridizing with a third barcoded oligonucleotide (e.g., ananalyte capture agent) coupled to an antibody that is bound to thesurface of the partitioned cell. The third barcoded oligonucleotideincludes a UMI barcode sequence that uniquely identifies the antibody(and thus, the particular cell surface feature to which it is bound).

In this example, the first and second barcoded oligonucleotides includethe same spatial barcode sequence (e.g., the first and second barcodesequences are the same), which permits downstream association ofbarcoded nucleic acids with the partition. In some embodiments, however,the first and second barcode sequences are different.

The partition also includes lysis agents that aid in releasing nucleicacids from the cell and can also include an agent (e.g., a reducingagent) that can degrade the bead and/or break a covalent linkage betweenthe barcoded oligonucleotides and the bead, releasing them into thepartition. The first type of released barcoded oligonucleotide canhybridize with mRNA released from the cell and the second type ofreleased barcoded oligonucleotide can hybridize with the third type ofbarcoded oligonucleotide, forming barcoded constructs.

The first type of barcoded construct includes a spatial barcode sequencecorresponding to the first barcode sequence from the bead and a sequencecorresponding to the UMI barcode sequence from the first type ofoligonucleotide, which identifies cell transcripts. The second type ofbarcoded construct includes a spatial barcode sequence corresponding tothe second barcode sequence from the second type of oligonucleotide, anda UMI barcode sequence corresponding to the third type ofoligonucleotide (e.g., the analyte capture agent) and used to identifythe cell surface feature. The barcoded constructs can then bereleased/removed from the partition and, in some embodiments, furtherprocessed to add any additional sequences. The resulting constructs arethen sequenced, sequencing data processed, and the results used tocharacterize the mRNA and cell surface feature of the cell.

The foregoing discussion involves two specific examples of beads witholigonucleotides for analyzing two different analytes within apartition. More generally, beads that are partitioned can have any ofthe structures described previously, and can include any of thedescribed combinations of oligonucleotides for analysis of two or more(e.g., three or more, four or more, five or more, six or more, eight ormore, ten or more, 12 or more, 15 or more, 20 or more, 25 or more, 30 ormore, 40 or more, 50 or more) different types of analytes within apartition. Examples of beads with combinations of different types ofoligonucleotides (e.g., capture probes) for concurrently analyzingdifferent combinations of analytes within partitions include, but arenot limited to: (a) genomic DNA and cell surface features (e.g., usingthe analyte capture agents described herein); (b) mRNA and a lineagetracing construct; (c) mRNA and cell methylation status; (d) mRNA andaccessible chromatin (e.g., ATAC-seq, DNase-seq, and/or MNase-seq); (e)mRNA and cell surface or intracellular proteins and/or metabolites; (f)a barcoded analyte capture agent (e.g., the MHC multimers describedherein) and a V(D)J sequence of an immune cell receptor (e.g., T-cellreceptor); and (g) mRNA and a perturbation agent (e.g., a CRISPRcrRNA/sgRNA, TALEN, zinc finger nuclease, and/or antisenseoligonucleotide as described herein).

Additionally, in some embodiments, the unaggregated cell ordisaggregated cells introduced and processed within partitions ordroplets as described herein, can be removed from the partition,contacted with a spatial array, and spatially barcoded according tomethods described herein. For example, single cells of an unaggregatedcell sample can be partitioned into partitions or droplets as describedherein. The partitions or droplets can include reagents to permeabilizea cell, barcode targeted cellular analyte(s) with a cellular barcode,and amplify the barcoded analytes. The partitions or droplets can becontacted with any of the spatial arrays described herein. In someembodiments, the partition can be dissolved, such that the contents ofthe partition are placed in contact with the capture probes of thespatial array. The capture probes of the spatial array can then capturetarget analytes from the ruptured partitions or the droplets, andprocessed by the spatial workflows described herein.

(G) Analysis of Captured Analytes Removal of Sample From Array

In some embodiments, after contacting a biological sample with asubstrate that includes capture probes, a removal step can optionally beperformed to remove all or a portion of the biological sample from thesubstrate. In some embodiments, the removal step includes enzymaticand/or chemical degradation of cells of the biological sample. Forexample, the removal step can include treating the biological samplewith an enzyme (e.g., a proteinase, e.g., proteinase K) to remove atleast a portion of the biological sample from the substrate. In someembodiments, the removal step can include ablation of the tissue (e.g.,laser ablation).

In some embodiments, provided herein are methods for spatially detectingan analyte (e.g., detecting the location of an analyte, e.g., abiological analyte) from a biological sample (e.g., present in abiological sample), the method comprising: (a) optionally stainingand/or imaging a biological sample on a substrate; (b) permeabilizing(e.g., providing a solution comprising a permeabilization reagent to)the biological sample on the substrate; (c) contacting the biologicalsample with an array comprising a plurality of capture probes, wherein acapture probe of the plurality captures the biological analyte; and (d)analyzing the captured biological analyte, thereby spatially detectingthe biological analyte; wherein the biological sample is fully orpartially removed from the substrate.

In some embodiments, a biological sample is not removed from thesubstrate. For example, the biological sample is not removed from thesubstrate prior to releasing a capture probe (e.g., a capture probebound to an analyte) from the substrate. In some embodiments, suchreleasing comprises cleavage of the capture probe from the substrate(e.g., via a cleavage domain). In some embodiments, such releasing doesnot comprise releasing the capture probe from the substrate (e.g., acopy of the capture probe bound to an analyte can be made and the copycan be released from the substrate, e.g., via denaturation). In someembodiments, the biological sample is not removed from the substrateprior to analysis of an analyte bound to a capture probe after it isreleased from the substrate. In some embodiments, the biological sampleremains on the substrate during removal of a capture probe from thesubstrate and/or analysis of an analyte bound to the capture probe afterit is released from the substrate. In some embodiments, analysis of ananalyte bound to capture probe from the substrate can be performedwithout subjecting the biological sample to enzymatic and/or chemicaldegradation of the cells (e.g., permeabilized cells) or ablation of thetissue (e.g., laser ablation).

In some embodiments, at least a portion of the biological sample is notremoved from the substrate. For example, a portion of the biologicalsample can remain on the substrate prior to releasing a capture probe(e.g., a capture prove bound to an analyte) from the substrate and/oranalyzing an analyte bound to a capture probe released from thesubstrate. In some embodiments, at least a portion of the biologicalsample is not subjected to enzymatic and/or chemical degradation of thecells (e.g., permeabilized cells) or ablation of the tissue (e.g., laserablation) prior to analysis of an analyte bound to a capture probe fromthe substrate.

In some embodiments, provided herein are methods for spatially detectingan analyte (e.g., detecting the location of an analyte, e.g., abiological analyte) from a biological sample (e.g., present in abiological sample) that include: (a) optionally staining and/or imaginga biological sample on a substrate; (b) permeabilizing (e.g., providinga solution comprising a permeabilization reagent to) the biologicalsample on the substrate; (c) contacting the biological sample with anarray comprising a plurality of capture probes, wherein a capture probeof the plurality captures the biological analyte; and (d) analyzing thecaptured biological analyte, thereby spatially detecting the biologicalanalyte; where the biological sample is not removed from the substrate.

In some embodiments, provided herein are methods for spatially detectinga biological analyte of interest from a biological sample that include:(a) staining and imaging a biological sample on a substrate; (b)providing a solution comprising a permeabilization reagent to thebiological sample on the substrate; (c) contacting the biological samplewith an array on a substrate, wherein the array comprises one or morecapture probe pluralities thereby allowing the one or more pluralitiesof capture probes to capture the biological analyte of interest; and (d)analyzing the captured biological analyte, thereby spatially detectingthe biological analyte of interest; where the biological sample is notremoved from the substrate.

In some embodiments, the method further includes selecting a region ofinterest in the biological sample to subject to spatial transcriptomicanalysis. In some embodiments, one or more of the one or more captureprobes include a capture domain. In some embodiments, one or more of theone or more capture probe pluralities comprise a unique molecularidentifier (UMI). In some embodiments, one or more of the one or morecapture probe pluralities comprise a cleavage domain. In someembodiments, the cleavage domain comprises a sequence recognized andcleaved by a uracil-DNA glycosylase, apurinic/apyrimidinic (AP)endonuclease (APE1), U uracil-specific excision reagent (USER), and/oran endonuclease VIII. In some embodiments, one or more capture probes donot comprise a cleavage domain and is not cleaved from the array.

A set of experiments performed determined methods that did not removethe biological sample from the substrate yielded higher qualitysequencing data, higher median genes per cell, and higher median UMIcounts per cell compared to a similar methods where the biologicalsample was removed from the substrate (data not shown).

In some embodiments, a capture probe can be extended. For example,extending a capture probe can includes generating cDNA from a captured(hybridized) RNA. This process involves synthesis of a complementarystrand of the hybridized nucleic acid, e.g., generating cDNA based onthe captured RNA template (the RNA hybridized to the capture domain ofthe capture probe). Thus, in an initial step of extending a captureprobe, e.g., the cDNA generation, the captured (hybridized) nucleicacid, e.g., RNA, acts as a template for the extension, e.g., reversetranscription, step.

In some embodiments, the capture probe is extended using reversetranscription. For example, reverse transcription includes synthesizingcDNA (complementary or copy DNA) from RNA, e.g., (messenger RNA), usinga reverse transcriptase. In some embodiments, reverse transcription isperformed while the tissue is still in place, generating an analytelibrary, where the analyte library includes the spatial barcodes fromthe adjacent capture probes. In some embodiments, the capture probe isextended using one or more DNA polymerases.

In some embodiments, a capture domain of a capture probe includes aprimer for producing the complementary strand of a nucleic acidhybridized to the capture probe, e.g., a primer for DNA polymeraseand/or reverse transcription. The nucleic acid, e.g., DNA and/or cDNA,molecules generated by the extension reaction incorporate the sequenceof the capture probe. The extension of the capture probe, e.g., a DNApolymerase and/or reverse transcription reaction, can be performed usinga variety of suitable enzymes and protocols.

In some embodiments, a full-length DNA, e.g. cDNA, molecule isgenerated. In some embodiments, a “full-length” DNA molecule refers tothe whole of the captured nucleic acid molecule. However, if the nucleicacid, e.g. RNA, was partially degraded in the tissue sample, then thecaptured nucleic acid molecules will not be the same length as theinitial RNA in the tissue sample. In some embodiments, the 3′ end of theextended probes, e.g., first strand cDNA molecules, is modified. Forexample, a linker or adaptor can be ligated to the 3′ end of theextended probes. This can be achieved using single stranded ligationenzymes such as T4 RNA ligase or Circligase™ (available from EpicentreBiotechnologies, Madison, WI). In some embodiments, template switchingoligonucleotides are used to extend cDNA in order to generate afull-length cDNA (or as close to a full-length cDNA as possible). Insome embodiments, a second strand synthesis helper probe (a partiallydouble stranded DNA molecule capable of hybridizing to the 3′ end of theextended capture probe), can be ligated to the 3′ end of the extendedprobe, e.g., first strand cDNA, molecule using a double strandedligation enzyme such as T4 DNA ligase. Other enzymes appropriate for theligation step are known in the art and include, e.g., Tth DNA ligase,Taq DNA ligase, Thermococcus sp. (strain 9°N) DNA ligase (9°N™ DNAligase, New England Biolabs), Ampligase™ (available from EpicentreBiotechnologies, Madison, WI), and SplintR (available from New EnglandBiolabs, Ipswich, MA). In some embodiments, a polynucleotide tail, e.g.,a poly(A) tail, is incorporated at the 3′ end of the extended probemolecules. In some embodiments, the polynucleotide tail is incorporatedusing a terminal transferase active enzyme.

In some embodiments, double-stranded extended capture probes are treatedto remove any unextended capture probes prior to amplification and/oranalysis, e.g. sequence analysis. This can be achieved by a variety ofmethods, e.g., using an enzyme to degrade the unextended probes, such asan exonuclease enzyme, or purification columns.

In some embodiments, extended capture probes are amplified to yieldquantities that are sufficient for analysis, e.g., via DNA sequencing.In some embodiments, the first strand of the extended capture probes(e.g., DNA and/or cDNA molecules) acts as a template for theamplification reaction (e.g., a polymerase chain reaction).

In some embodiments, the amplification reaction incorporates an affinitygroup onto the extended capture probe (e.g., RNA-cDNA hybrid) using aprimer including the affinity group. In some embodiments, the primerincludes an affinity group and the extended capture probes includes theaffinity group. The affinity group can correspond to any of the affinitygroups described previously.

In some embodiments, the extended capture probes including the affinitygroup can be coupled to a substrate specific for the affinity group. Insome embodiments, the substrate can include an antibody or antibodyfragment. In some embodiments, the substrate includes avidin orstreptavidin and the affinity group includes biotin. In someembodiments, the substrate includes maltose and the affinity groupincludes maltose-binding protein. In some embodiments, the substrateincludes maltose-binding protein and the affinity group includesmaltose. In some embodiments, amplifying the extended capture probes canfunction to release the extended probes from the surface of thesubstrate, insofar as copies of the extended probes are not immobilizedon the substrate.

In some embodiments, the extended capture probe or complement oramplicon thereof is released. The step of releasing the extended captureprobe or complement or amplicon thereof from the surface of thesubstrate can be achieved in a number of ways. In some embodiments, anextended capture probe or a complement thereof is released from thearray by nucleic acid cleavage and/or by denaturation (e.g. by heatingto denature a double-stranded molecule).

In some embodiments, the extended capture probe or complement oramplicon thereof is released from the surface of the substrate (e.g.,array) by physical means. For example, where the extended capture probeis indirectly immobilized on the array substrate, e.g. via hybridizationto a surface probe, it can be sufficient to disrupt the interactionbetween the extended capture probe and the surface probe. Methods fordisrupting the interaction between nucleic acid molecules includedenaturing double stranded nucleic acid molecules art. A straightforwardmethod for releasing the DNA molecules (i.e., of stripping the array ofthe extended probes) is to use a solution that interferes with thehydrogen bonds of the double stranded molecules. In some embodiments,the extended capture probe is released by applying heated water such aswater or buffer of at least 85° C., e.g., at least 90, 91, 92, 93, 94,95, 96, 97, 98, or 99° C. In some embodiments, a solution includingsalts, surfactants, etc. that can further destabilize the interactionbetween the nucleic acid molecules is added to release the extendedcapture probe from the substrate.

In some embodiments, where the extended capture probe includes acleavage domain, the extended capture probe is released from the surfaceof the substrate by cleavage. For example, the cleavage domain of theextended capture probe can be cleaved by any of the methods describedherein. In some embodiments, the extended capture probe is released fromthe surface of the substrate, e.g., via cleavage of a cleavage domain inthe extended capture probe, prior to the step of amplifying the extendedcapture probe.

Capture probes can optionally include a “cleavage domain,” where one ormore segments or regions of the capture probe (e.g., spatial barcodesand/or UMIs) can be releasably, cleavably, or reversibly attached to afeature, or some other substrate, so that spatial barcodes and/or UMIscan be released or be releasable through cleavage of a linkage betweenthe capture probe and the feature, or released through degradation ofthe underlying support, allowing the spatial barcode(s) and/or UMI(s) ofthe cleaved capture probe to be accessed or be accessible by otherreagents, or both.

In some embodiments, the capture probe is linked, via a disulfide bond,to a feature. In some embodiments, the capture probe is linked to afeature via a propylene group (e.g., Spacer C3). A reducing agent can beadded to break the various disulfide bonds, resulting in release of thecapture probe including the spatial barcode sequence. In anotherexample, heating can also result in degradation and release of theattached capture probe. In some embodiments, the heating is done bylaser (e.g., laser ablation) and features at specific locations can bedegraded. In addition to thermally cleavable bonds, disulfide bonds,photo-sensitive bonds, and UV sensitive bonds, other non-limitingexamples of labile bonds that can be coupled to a capture probe (i.e.,spatial barcode) include an ester linkage (e.g., cleavable with an acid,a base, or hydroxylamine), a vicinal diol linkage (e.g., cleavable viasodium periodate), a Diels-Alder linkage (e.g., cleavable via heat), asulfone linkage (e.g., cleavable via a base), a silyl ether linkage(e.g., cleavable via an acid), a glycosidic linkage (e.g., cleavable viaan amylase), a peptide linkage (e.g., cleavable via a protease), or aphosphodiester linkage (e.g., cleavable via a nuclease (e.g., DNAase)).

In some embodiments, the cleavage domain includes a sequence that isrecognized by one or more enzymes capable of cleaving a nucleic acidmolecule, e.g., capable of breaking the phosphodiester linkage betweentwo or more nucleotides. A bond can be cleavable via other nucleic acidmolecule targeting enzymes, such as restriction enzymes (e.g.,restriction endonucleases). For example, the cleavage domain can includea restriction endonuclease (restriction enzyme) recognition sequence.Restriction enzymes cut double-stranded or single stranded DNA atspecific recognition nucleotide sequences known as restriction sites. Insome embodiments, a rare-cutting restriction enzyme, i.e., enzymes witha long recognition site (at least 8 base pairs in length), is used toreduce the possibility of cleaving elsewhere in the capture probe.

In some embodiments, the cleavage domain includes a poly(U) sequencewhich can be cleaved by a mixture of Uracil DNA glycosylase (UDG) andthe DNA glycosylase-lyase Endonuclease VIII, commercially known as theUSER™ enzyme. In some embodiments, the cleavage domain can be a singleU. In some embodiments, the cleavage domain can be an abasic site thatcan be cleaved with an abasic site-specific endonuclease (e.g.,Endonucleoase IV or Endonuclease VIII). Releasable capture probes can beavailable for reaction once released. Thus, for example, an activatablecapture probe can be activated by releasing the capture probes from afeature.

In some embodiments, the cleavage domain of the capture probe is anucleotide sequence within the capture probe that is cleavedspecifically, e.g., physically by light or heat, chemically orenzymatically. The location of the cleavage domain within the captureprobe will depend on whether or not the capture probe is immobilized onthe substrate such that it has a free 3′ end capable of functioning asan extension primer (e.g. by its 5′ or 3′ end). For example, if thecapture probe is immobilized by its 5′ end, the cleavage domain will belocated 5′ to the spatial barcode and/or UMI, and cleavage of saiddomain results in the release of part of the capture probe including thespatial barcode and/or UMI and the sequence 3′ to the spatial barcode,and optionally part of the cleavage domain, from a feature.Alternatively, if the capture probe is immobilized by its 3′ end, thecleavage domain will be located 3′ to the capture domain (and spatialbarcode) and cleavage of said domain results in the release of part ofthe capture probe including the spatial barcode and the sequence 3′ tothe spatial barcode from a feature. In some embodiments, cleavageresults in partial removal of the cleavage domain. In some embodiments,cleavage results in complete removal of the cleavage domain,particularly when the capture probes are immobilized via their 3′ end asthe presence of a part of the cleavage domain can interfere with thehybridization of the capture domain and the target nucleic acid and/orits subsequent extension.

In some embodiments, where the capture probe is immobilized to thesubstrate indirectly, e.g., via a surface probe defined below, thecleavage domain includes one or more mismatch nucleotides, so that thecomplementary parts of the surface probe and the capture probe are not100% complementary (for example, the number of mismatched base pairs canone, two, or three base pairs). Such a mismatch is recognized, e.g., bythe MutY and T7 endonuclease I enzymes, which results in cleavage of thenucleic acid molecule at the position of the mismatch.

In some embodiments, where the capture probe is immobilized to thefeature indirectly, e.g., via a surface probe, the cleavage domainincludes a nickase recognition site or sequence. In this respect,nickase enzymes cleave only one strand in a nucleic acid duplex.Nickases are endonucleases which cleave only a single strand of a DNAduplex. Thus, the cleavage domain can include a nickase recognition siteclose to the 5′ end of the surface probe (and/or the 5′ end of thecapture probe) such that cleavage of the surface probe or capture probedestabilizes the duplex between the surface probe and capture probethereby releasing the capture probe) from the feature.

Nickase enzymes can also be used in some embodiments where the captureprobe is immobilized to the feature directly. For example, the substratecan be contacted with a nucleic acid molecule that hybridizes to thecleavage domain of the capture probe to provide or reconstitute anickase recognition site, e.g., a cleavage helper probe. Thus, contactwith a nickase enzyme will result in cleavage of the cleavage domainthereby releasing the capture probe from the feature. Such cleavagehelper probes can also be used to provide or reconstitute cleavagerecognition sites for other cleavage enzymes, e.g., restriction enzymes.

Some nickases introduce single-stranded nicks only at particular siteson a DNA molecule, by binding to and recognizing a particular nucleotiderecognition sequence. A number of naturally-occurring nickases have beendiscovered, of which at present the sequence recognition properties havebeen determined for at least four. Nickases are described in U.S. Pat.No. 6,867,028, which is herein incorporated by reference in itsentirety. In general, any suitable nickase can be used to bind to acomplementary nickase recognition site of a cleavage domain. Followinguse, the nickase enzyme can be removed from the assay or inactivatedfollowing release of the capture probes to prevent unwanted cleavage ofthe capture probes.

In some embodiments, a cleavage domain for separating spatial barcodesfrom a feature is absent from the capture probe. For example, asubstrate having a capture probe lacking a cleavage domain can be usedfor spatial analysis (see, e.g., corresponding substrates and probesdescribed Macosko et al., (2015) Cell 161, 1202-1214, the entirecontents of which are incorporated herein by reference.

In some embodiments, the region of the capture probe corresponding tothe cleavage domain can be used for some other function. For example, anadditional region for nucleic acid extension or amplification can beincluded where the cleavage domain would normally be positioned. In suchembodiments, the region can supplement the functional domain or evenexist as an additional functional domain. In some embodiments, thecleavage domain is present but its use is optional.

After analytes from the sample have hybridized or otherwise beenassociated with capture probes, analyte capture agents, or otherbarcoded oligonucleotide sequences according to any of the methodsdescribed above in connection with the general spatial cell-basedanalytical methodology, the barcoded constructs that result fromhybridization/association are analyzed via sequencing to identify theanalytes.

In some embodiments, where a sample is barcoded directly viahybridization with capture probes or analyte capture agents hybridized,bound, or associated with either the cell surface, or introduced intothe cell, as described above, sequencing can be performed on the intactsample. Alternatively, if the barcoded sample has been separated intofragments, cell groups, or individual cells, as described above,sequencing can be performed on individual fragments, cell groups, orcells. For analytes that have been barcoded via partitioning with beads,as described above, individual analytes (e.g., cells, or cellularcontents following lysis of cells) can be extracted from the partitionsby breaking the partitions, and then analyzed by sequencing to identifythe analytes.

A wide variety of different sequencing methods can be used to analyzebarcoded analyte constructs. In general, sequenced polynucleotides canbe, for example, nucleic acid molecules such as deoxyribonucleic acid(DNA) or ribonucleic acid (RNA), including variants or derivativesthereof (e.g., single stranded DNA or DNA/RNA hybrids, and nucleic acidmolecules with a nucleotide analog).

Sequencing of polynucleotides can be performed by various commercialsystems. More generally, sequencing can be performed using nucleic acidamplification, polymerase chain reaction (PCR) (e.g., digital PCR anddroplet digital PCR (ddPCR), quantitative PCR, real time PCR, multiplexPCR, PCR-based singleplex methods, emulsion PCR), and/or isothermalamplification.

Other examples of methods for sequencing genetic material include, butare not limited to, DNA hybridization methods (e.g., Southern blotting),restriction enzyme digestion methods, Sanger sequencing methods,next-generation sequencing methods (e.g., single-molecule real-timesequencing, nanopore sequencing, and Polony sequencing), ligationmethods, and microarray methods. Additional examples of sequencingmethods that can be used include targeted sequencing, single moleculereal-time sequencing, exon sequencing, electron microscopy-basedsequencing, panel sequencing, transistor-mediated sequencing, directsequencing, random shotgun sequencing, Sanger dideoxy terminationsequencing, whole-genome sequencing, sequencing by hybridization,pyrosequencing, capillary electrophoresis, gel electrophoresis, duplexsequencing, cycle sequencing, single-base extension sequencing,solid-phase sequencing, high-throughput sequencing, massively parallelsignature sequencing, co-amplification at lower denaturationtemperature-PCR (COLD-PCR), sequencing by reversible dye terminator,paired-end sequencing, near-term sequencing, exonuclease sequencing,sequencing by ligation, short-read sequencing, single-moleculesequencing, sequencing-by-synthesis, real-time sequencing,reverse-terminator sequencing, nanopore sequencing, 454 sequencing,Solexa Genome Analyzer sequencing, SOLiD™ sequencing, MS-PET sequencing,and any combinations thereof.

Sequence analysis of the nucleic acid molecules (including barcodednucleic acid molecules or derivatives thereof) can be direct orindirect. Thus, the sequence analysis substrate (which can be viewed asthe molecule which is subjected to the sequence analysis step orprocess) can directly be the barcoded nucleic acid molecule or it can bea molecule which is derived therefrom (e.g., a complement thereof).Thus, for example, in the sequence analysis step of a sequencingreaction, the sequencing template can be the barcoded nucleic acidmolecule or it can be a molecule derived therefrom. For example, a firstand/or second strand DNA molecule can be directly subjected to sequenceanalysis (e.g. sequencing), i.e., can directly take part in the sequenceanalysis reaction or process (e.g. the sequencing reaction or sequencingprocess, or be the molecule which is sequenced or otherwise identified).Alternatively, the barcoded nucleic acid molecule can be subjected to astep of second strand synthesis or amplification before sequenceanalysis (e.g. sequencing or identification by another technique). Thesequence analysis substrate (e.g., template) can thus be an amplicon ora second strand of a barcoded nucleic acid molecule.

In some embodiments, both strands of a double stranded molecule can besubjected to sequence analysis (e.g., sequenced). In some embodiments,single stranded molecules (e.g. barcoded nucleic acid molecules) can beanalyzed (e.g. sequenced). To perform single molecule sequencing, thenucleic acid strand can be modified at the 3′ end.

Massively parallel sequencing techniques can be used for sequencingnucleic acids, as described above. In one embodiment, a massivelyparallel sequencing technique can be based on reversibledye-terminators. As an example, DNA molecules are first attached toprimers on, e.g., a glass or silicon substrate, and amplified so thatlocal clonal colonies are formed (bridge amplification). Four types ofddNTPs are added, and non-incorporated nucleotides are washed away.Unlike pyrosequencing, the DNA is only extended one nucleotide at a timedue to a blocking group (e.g., 3′ blocking group present on the sugarmoiety of the ddNTP). A detector acquires images of the fluorescentlylabelled nucleotides, and then the dye along with the terminal 3′blocking group is chemically removed from the DNA, as a precursor to asubsequent cycle. This process can be repeated until the requiredsequence data is obtained.

As another example, massively parallel pyrosequencing techniques canalso be used for sequencing nucleic acids. In pyrosequencing, thenucleic acid is amplified inside water droplets in an oil solution(emulsion PCR), with each droplet containing a single nucleic acidtemplate attached to a single primer-coated bead that then forms aclonal colony. The sequencing system contains many picolitre-volumewells each containing a single bead and sequencing enzymes.Pyrosequencing uses luciferase to generate light for detection of theindividual nucleotides added to the nascent nucleic acid and thecombined data are used to generate sequence reads.

As another example application of pyrosequencing, released PPi can bedetected by being immediately converted to adenosine triphosphate (ATP)by ATP sulfurylase, and the level of ATP generated can be detected vialuciferase-produced photons, such as described in Ronaghi, et al., Anal.Biochem. 242(1), 84-9 (1996); Ronaghi, Genome Res. 11(1), 3-11 (2001);Ronaghi et al. Science 281 (5375), 363 (1998); and U.S. Pat. Nos.6,210,891, 6,258,568, and 6,274,320, the entire contents of each ofwhich are incorporated herein by reference.

In some embodiments, sequencing is performed by detection of hydrogenions that are released during the polymerization of DNA. A microwellcontaining a template DNA strand to be sequenced can be flooded with asingle type of nucleotide. If the introduced nucleotide is complementaryto the leading template nucleotide, it is incorporated into the growingcomplementary strand. This causes the release of a hydrogen ion thattriggers a hypersensitive ion sensor, which indicates that a reactionhas occurred. If homopolymer repeats are present in the templatesequence, multiple nucleotides will be incorporated in a single cycle.This leads to a corresponding number of released hydrogen ions and aproportionally higher electronic signal.

In some embodiments, sequencing can be performed in-situ. In-situsequencing methods are particularly useful, for example, when thebiological sample remains intact after analytes on the sample surface(e.g., cell surface analytes) or within the sample (e.g., intracellularanalytes) have been barcoded. In-situ sequencing typically involvesincorporation of a labeled nucleotide (e.g., fluorescently labeledmononucleotides or dinucleotides) in a sequential, template-dependentmanner or hybridization of a labeled primer (e.g., a labeled randomhexamer) to a nucleic acid template such that the identities (i.e.,nucleotide sequence) of the incorporated nucleotides or labeled primerextension products can be determined, and consequently, the nucleotidesequence of the corresponding template nucleic acid. Aspects of in-situsequencing are described, for example, in Mitra et al., (2003) Anal.Biochem. 320, 55-65, and Lee et al., (2014) Science, 343(6177),1360-1363, the entire contents of each of which are incorporated hereinby reference.

In addition, examples of methods and systems for performing in-situsequencing are described in PCT Pat. Application Publication Nos.WO2014/163886, WO2018/045181, WO2018/045186, and in U.S. Pat. Nos.10,138,509 and 10,179,932, the entire contents of each of which areincorporated herein by reference. Example techniques for in-situsequencing include, but are not limited to, STARmap (described forexample in Wang et al., (2018) Science, 361(6499) 5691), MERFISH(described for example in Moffitt, (2016) Methods in Enzymology, 572,1-49), and FISSEQ (described for example in U.S. Pat. ApplicationPublication No. 2019/0032121). The entire contents of each of theforegoing references are incorporated herein by reference.

For analytes that have been barcoded via partitioning, barcoded nucleicacid molecules or derivatives thereof (e.g., barcoded nucleic acidmolecules to which one or more functional sequences have been added, orfrom which one or more features have been removed) can be pooled andprocessed together for subsequent analysis such as sequencing on highthroughput sequencers. Processing with pooling can be implemented usingbarcode sequences. For example, barcoded nucleic acid molecules of agiven partition can have the same barcode, which is different frombarcodes of other spatial partitions. Alternatively, barcoded nucleicacid molecules of different partitions can be processed separately forsubsequent analysis (e.g., sequencing).

In some embodiments, where capture probes do not contain a spatialbarcode, the spatial barcode can be added after the capture probecaptures analytes from a biological sample and before analysis of theanalytes. When a spatial barcode is added after an analyte is captured,the barcode can be added after amplification of the analyte (e.g.,reverse transcription and polymerase amplification of RNA). In someembodiments, analyte analysis uses direct sequencing of one or morecaptured analytes, such as direct sequencing of hybridized RNA. In someembodiments, direct sequencing is performed after reverse transcriptionof hybridized RNA. In some embodiments direct sequencing is performedafter amplification of reverse transcription of hybridized RNA.

In some embodiments, direct sequencing of captured RNA is performed bysequencing-by-synthesis (SBS). In some embodiments, a sequencing primeris complementary to a sequence in one or more of the domains of acapture probe (e.g., functional domain). In such embodiments,sequencing-by-synthesis can include reverse transcription and/oramplification in order to generate a template sequence (e.g., functionaldomain) from which a primer sequence can bind.

SBS can involve hybridizing an appropriate primer, sometimes referred toas a sequencing primer, with the nucleic acid template to be sequenced,extending the primer, and detecting the nucleotides used to extend theprimer. Preferably, the nucleic acid used to extend the primer isdetected before a further nucleotide is added to the growing nucleicacid chain, thus allowing base-by-base in situ nucleic acid sequencing.The detection of incorporated nucleotides is facilitated by includingone or more labelled nucleotides in the primer extension reaction. Toallow the hybridization of an appropriate sequencing primer to thenucleic acid template to be sequenced, the nucleic acid template shouldnormally be in a single stranded form. If the nucleic acid templatesmaking up the nucleic acid spots are present in a double stranded formthese can be processed to provide single stranded nucleic acid templatesusing methods well known in the art, for example by denaturation,cleavage etc. The sequencing primers which are hybridized to the nucleicacid template and used for primer extension are preferably shortoligonucleotides, for example, 15 to 25 nucleotides in length. Thesequencing primers can be provided in solution or in an immobilizedform. Once the sequencing primer has been annealed to the nucleic acidtemplate to be sequenced by subjecting the nucleic acid template andsequencing primer to appropriate conditions, primer extension is carriedout, for example using a nucleic acid polymerase and a supply ofnucleotides, at least some of which are provided in a labelled form, andconditions suitable for primer extension if a suitable nucleotide isprovided.

Preferably after each primer extension step, a washing step is includedin order to remove unincorporated nucleotides which can interfere withsubsequent steps. Once the primer extension step has been carried out,the nucleic acid colony is monitored to determine whether a labellednucleotide has been incorporated into an extended primer. The primerextension step can then be repeated to determine the next and subsequentnucleotides incorporated into an extended primer. If the sequence beingdetermined is unknown, the nucleotides applied to a given colony areusually applied in a chosen order which is then repeated throughout theanalysis, for example dATP, dTTP, dCTP, dGTP.

SBS techniques which can be used are described for example, but notlimited to, those in U.S. Pat. App. Pub. No. 2007/0166705, U.S. Pat.App. Pub. No. 2006/0188901, U.S. Pat. 7,057,026, U.S. Pat. App. Pub. No.2006/0240439, U.S. Pat. App. Pub. No. 2006/0281109, PCT Pat. App. Pub.No. WO 05/065814, U.S. Pat. App. Pub. No. 2005/0100900, PCT Pat. App.Pub. No. WO 06/064199, PCT Pat. App. Pub. No. WO07/010,251, U.S. Pat.App. Pub. No. 2012/0270305, U.S. Pat. App. Pub. No. 2013/0260372, andU.S. Pat. App. Pub. No. 2013/0079232, the entire contents of each ofwhich are incorporated herein by reference.

In some embodiments, direct sequencing of captured RNA is performed bysequential fluorescence hybridization (e.g., sequencing byhybridization). In some embodiments, a hybridization reaction where RNAis hybridized to a capture probe is performed in situ. In someembodiments, captured RNA is not amplified prior to hybridization with asequencing probe. In some embodiments, RNA is amplified prior tohybridization with sequencing probes (e.g., reverse transcription tocDNA and amplification of cDNA). In some embodiments, amplification isperformed using single-molecule hybridization chain reaction. In someembodiments, amplification is performed using rolling chainamplification.

Sequential fluorescence hybridization can involve sequentialhybridization of probes including degenerate primer sequences and adetectable label. A degenerate primer sequence is a shortoligonucleotide sequence which is capable of hybridizing to any nucleicacid fragment independent of the sequence of said nucleic acid fragment.For example, such a method could include the steps of: (a) providing amixture including four probes, each of which includes either A, C, G, orT at the 5′-terminus, further including degenerate nucleotide sequenceof 5 to 11 nucleotides in length, and further including a functionaldomain (e.g., fluorescent molecule) that is distinct for probes with A,C, G, or T at the 5′-terminus; (b) associating the probes of step (a) tothe target polynucleotide sequences, whose sequence needs will bedetermined by this method; (c) measuring the activities of the fourfunctional domains and recording the relative spatial location of theactivities; (d) removing the reagents from steps (a)-(b) from the targetpolynucleotide sequences; and repeating steps (a)-(d) for n cycles,until the nucleotide sequence of the spatial domain for each bead isdetermined, with modification that the oligonucleotides used in step (a)are complementary to part of the target polynucleotide sequences and thepositions 1 through n flanking the part of the sequences. Because thebarcode sequences are different, in some embodiments, these additionalflanking sequences are degenerate sequences. The fluorescent signal fromeach spot on the array for cycles 1 through n can be used to determinethe sequence of the target polynucleotide sequences.

In some embodiments, direct sequencing of captured RNA using sequentialfluorescence hybridization is performed in vitro. In some embodiments,captured RNA is amplified prior to hybridization with a sequencing probe(e.g., reverse transcription to cDNA and amplification of cDNA). In someembodiments, a capture probe containing captured RNA is exposed to thesequencing probe targeting coding regions of RNA. In some embodiments,one or more sequencing probes are targeted to each coding region. Insome embodiments, the sequencing probe is designed to hybridize withsequencing reagents (e.g., a dye-labeled readout oligonucleotides). Asequencing probe can then hybridize with sequencing reagents. In someembodiments, output from the sequencing reaction is imaged. In someembodiments, a specific sequence of cDNA is resolved from an image of asequencing reaction. In some embodiments, reverse transcription ofcaptured RNA is performed prior to hybridization to the sequencingprobe. In some embodiments, the sequencing probe is designed to targetcomplementary sequences of the coding regions of RNA (e.g., targetingcDNA).

In some embodiments, a captured RNA is directly sequenced using ananopore-based method. In some embodiments, direct sequencing isperformed using nanopore direct RNA sequencing in which captured RNA istranslocated through a nanopore. A nanopore current can be recorded andconverted into a base sequence. In some embodiments, captured RNAremains attached to a substrate during nanopore sequencing. In someembodiments, captured RNA is released from the substrate prior tonanopore sequencing. In some embodiments, where the analyte of interestis a protein, direct sequencing of the protein can be performed usingnanopore-based methods. Examples of nanopore-based sequencing methodsthat can be used are described in Deamer et al., Trends Biotechnol. 18,14 7-151 (2000); Deamer et al., Acc. Chem. Res. 35:817-825 (2002); Li etal., Nat. Mater. 2:611-615 (2003); Soni et al., Clin. Chem. 53,1996-2001 (2007); Healy et al., Nanomed. 2, 459-481 (2007); Cockroft etal., J. Am. Chem. Soc. 130, 818-820 (2008); and in U.S. Pat. 7,001,792.The entire contents of each of the foregoing references are incorporatedherein by reference.

In some embodiments, direct sequencing of captured RNA is performedusing single molecule sequencing by ligation. Such techniques utilizeDNA ligase to incorporate oligonucleotides and identify theincorporation of such oligonucleotides. The oligonucleotides typicallyhave different labels that are correlated with the identity of aparticular nucleotide in a sequence to which the oligonucleotideshybridize. Aspects and features involved in sequencing by ligation aredescribed, for example, in Shendure et al. Science (2005), 309:1728-1732, and in U.S. Pat. Nos. 5,599,675; 5,750,341; 6,969,488;6,172,218; and 6,306,597, the entire contents of each of which areincorporated herein by reference.

In some embodiments, nucleic acid hybridization can be used forsequencing. These methods utilize labeled nucleic acid decoder probesthat are complementary to at least a portion of a barcode sequence.Multiplex decoding can be performed with pools of many different probeswith distinguishable labels. Non-limiting examples of nucleic acidhybridization sequencing are described for example in U.S. Pat. No.8,460,865, and in Gunderson et al., Genome Research 14:870-877 (2004),the entire contents of each of which are incorporated herein byreference.

In some embodiments, commercial high-throughput digital sequencingtechniques can be used to analyze barcode sequences, in which DNAtemplates are prepared for sequencing not one at a time, but in a bulkprocess, and where many sequences are read out preferably in parallel,or alternatively using an ultra-high throughput serial process thatitself may be parallelized. Examples of such techniques includeIllumina® sequencing (e.g., flow cell-based sequencing techniques),sequencing by synthesis using modified nucleotides (such ascommercialized in TruSeq™ and HiSeq™ technology by Illumina, Inc., SanDiego, CA), HeliScope™ by Helicos Biosciences Corporation, Cambridge,MA, and PacBio RS by Pacific Biosciences of California, Inc., MenloPark, CA), sequencing by ion detection technologies (Ion Torrent, Inc.,South San Francisco, CA), and sequencing of DNA nanoballs (CompleteGenomics, Inc., Mountain View, CA).

In some embodiments, detection of a proton released upon incorporationof a nucleotide into an extension product can be used in the methodsdescribed herein. For example, the sequencing methods and systemsdescribed in U.S. Pat. Application Publication Nos. 2009/0026082,2009/0127589, 2010/0137143, and 2010/0282617, can be used to directlysequence barcodes.

In some embodiments, real-time monitoring of DNA polymerase activity canbe used during sequencing. For example, nucleotide incorporations can bedetected through fluorescence resonance energy transfer (FRET), asdescribed for example in Levene et al., Science (2003), 299, 682-686,Lundquist et al., Opt. Lett. (2008), 33, 1026-1028, and Korlach et al.,Proc. Natl. Acad. Sci. USA (2008), 105, 1176-1181. The entire contentsof each of the foregoing references are incorporated herein by referenceherein.

In some embodiments, the methods described herein can be used to assessanalyte levels and/or expression in a cell or a biological sample overtime (e.g., before or after treatment with an agent or different stagesof differentiation). In some examples, the methods described herein canbe performed on multiple similar biological samples or cells obtainedfrom the subject at a different time points (e.g., before or aftertreatment with an agent, different stages of differentiation, differentstages of disease progression, different ages of the subject, or beforeor after development of resistance to an agent).

(H) Spatially Resolving Analyte Information

In some embodiments, a lookup table (LUT) can be used to associate oneproperty with another property of a feature. These properties include,e.g., locations, barcodes (e.g., nucleic acid barcode molecules),spatial barcodes, optical labels, molecular tags, and other properties.

In some embodiments, a lookup table can associate the plurality ofnucleic acid barcode molecules with the features. In some embodiments,the optical label of a feature can permit associating the feature withthe biological particle (e.g., cell or nuclei). The association of thefeature with the biological particle can further permit associating anucleic acid sequence of a nucleic acid molecule of the biologicalparticle to one or more physical properties of the biological particle(e.g., a type of a cell or a location of the cell). For example, basedon the relationship between the barcode and the optical label, theoptical label can be used to determine the location of a feature, thusassociating the location of the feature with the barcode sequence of thefeature. Subsequent analysis (e.g., sequencing) can associate thebarcode sequence and the analyte from the sample. Accordingly, based onthe relationship between the location and the barcode sequence, thelocation of the biological analyte can be determined (e.g., in aspecific type of cell, in a cell at a specific location of thebiological sample).

In some embodiments, the feature can have a plurality of nucleic acidbarcode molecules attached thereto. The plurality of nucleic acidbarcode molecules can include barcode sequences. The plurality ofnucleic acid molecules attached to a given feature can have the samebarcode sequences, or two or more different barcode sequences. Differentbarcode sequences can be used to provide improved spatial locationaccuracy.

As discussed above, analytes obtained from a sample, such as RNA, DNA,peptides, lipids, and proteins, can be further processed. In particular,the contents of individual cells from the sample can be provided withunique spatial barcode sequences such that, upon characterization of theanalytes, the analytes can be attributed as having been derived from thesame cell. More generally, spatial barcodes can be used to attributeanalytes to corresponding spatial locations in the sample. For example,hierarchical spatial positioning of multiple pluralities of spatialbarcodes can be used to identify and characterize analytes over aparticular spatial region of the sample. In some embodiments, thespatial region corresponds to a particular spatial region of interestpreviously identified, e.g., a particular structure of cytoarchitecturepreviously identified. In some embodiments, the spatial regioncorresponds to a small structure or group of cells that cannot be seenwith the naked eye. In some embodiments, a unique molecular identifiercan be used to identify and characterize analytes at a single celllevel.

The analyte can include a nucleic acid molecule, which can be barcodedwith a barcode sequence of a nucleic acid barcode molecule. In someembodiments, the barcoded analyte can be sequenced to obtain a nucleicacid sequence. In some embodiments, the nucleic acid sequence caninclude genetic information associate with the sample. The nucleic acidsequence can include the barcode sequence, or a complement thereof. Thebarcode sequence, or a complement thereof, of the nucleic acid sequencecan be electronically associated with the property (e.g., color and/orintensity) of the analyte using the LUT to identify the associatedfeature in an array.

In some embodiments, two- or three-dimensional spatial profiling of oneor more analytes present in a biological sample can be performed using aproximity capture reaction, which is a reaction that detects twoanalytes that are spatially close to each other and/or interacting witheach other. For example, a proximity capture reaction can be used todetect sequences of DNA that are close in space to each other, e.g., theDNA sequences can be within the same chromosome, but separated by about700 bp or less. As another example, a proximity capture reaction can beused to detect protein associations, e.g., two proteins that interactwith each other. A proximity capture reaction can be performed in situto detect two analytes that are spatially close to each other and/orinteracting with each other inside a cell. Non-limiting examples ofproximity capture reactions include DNA nanoscopy, DNA microscopy, andchromosome conformation capture methods. Chromosome conformation capture(3C) and derivative experimental procedures can be used to estimate thespatial proximity between different genomic elements. Non-limitingexamples of chromatin capture methods include chromosome conformationcapture (3-C), conformation capture-on-chip (4-C), 5-C, ChIA-PET, Hi-C,targeted chromatin capture (T2C). Examples of such methods aredescribed, for example, in Miele et al., Methods Mol Biol. (2009), 464,Simonis et al., Nat. Genet. (2006), 38(11): 1348-54, Raab et al., Embo.J. (2012), 31(2): 330-350, and Eagen et al., Trends Biochem. Sci. (2018)43(6): 469-478, the entire contents of each of which is incorporatedherein by reference.

In some embodiments, the proximity capture reaction includes proximityligation. In some embodiments, proximity ligation can include usingantibodies with attached DNA strands that can participate in ligation,replication, and sequence decoding reactions. For example, a proximityligation reaction can include oligonucleotides attached to pairs ofantibodies that can be joined by ligation if the antibodies have beenbrought in proximity to each oligonucleotide, e.g., by binding the sametarget protein (complex), and the DNA ligation products that form arethen used to template PCR amplification, as described for example inSoderberg et al., Methods. (2008), 45(3): 227-32, the entire contents ofwhich are incorporated herein by reference. In some embodiments,proximity ligation can include chromosome conformation capture methods.In some embodiments, the proximity capture reaction is performed onanalytes within about 400 nm distance (e.g., about 300 nm, about 200 nm,about 150 nm, about 100 nm, about 50 nm, about 25 nm, about 10 nm, orabout 5 nm) from each other. In general, proximity capture reactions canbe reversible or irreversible.

III. General Spatial Cell-Based Analytical Methodology (A) BarcodingBiological Sample

In some embodiments, provided herein are methods and materials forattaching and/or introducing a molecule (e.g., a nucleic acid molecule)having a barcode (e.g., a spatial barcode) to a biological sample (e.g.,to a cell in a biological sample) for use in spatial analysis. In someembodiments, a plurality of molecules (e.g., a plurality of nucleic acidmolecules) having a plurality of barcodes (e.g., a plurality of spatialbarcodes) are introduced to a biological sample (e.g., to a plurality ofcells in a biological sample) for use in spatial analysis.

FIG. 18 is a schematic diagram depicting cell tagging using eithercovalent conjugation of the analyte binding moiety to the cell surfaceor non-covalent interactions with cell membrane elements. FIG. 18 listsnon-exhaustive examples of a covalent analyte binding moiety/cellsurface interactions, including protein targeting, amine conjugationusing NHS chemistry, cyanuric chloride, thiol conjugation via maleimideaddition, as well as targeting glycoproteins/glycolipids expressed onthe cell surface via click chemistry. Non-exhaustive examples ofnon-covalent interactions with cell membrane elements include lipidmodified oligos, biocompatible anchor for cell membrane (oleyl-PEG-NHS),lipid modified positive neutral polymer, and antibody to membraneproteins. The cell tag can be used in combination with an analytecapture agent and cleavable or non-cleavable spatially-barcoded captureprobes for spatial and multiplexing applications.

In some embodiments, a plurality of molecules (e.g., a plurality ofnucleic acid molecules) having a plurality of barcodes (e.g., aplurality of spatial barcodes) are introduced to a biological sample(e.g., to a plurality of cells in a biological sample) for use inspatial analysis, wherein the plurality of molecules are introduced tothe biological sample in an arrayed format. In some embodiments, aplurality of molecules (e.g., a plurality of nucleic acid molecules)having a plurality of barcodes are provided on a substrate (e.g., any ofthe variety of substrates described herein) in any of the variety ofarrayed formats described herein, and the biological sample is contactedwith the molecules on the substrate such that the molecules areintroduced to the biological sample. In some embodiments, the moleculesthat are introduced to the biological sample are cleavably attached tothe substrate, and are cleaved from the substrate and released to thebiological sample when contacted with the biological sample. In someembodiments, the molecules that are introduced to the biological sampleare attached to the substrate covalently prior to cleavage. In someembodiments, the molecules that are introduced to the biological sampleare non-covalently attached to the substrate (e.g., via hybridization),and are released from the substrate to the biological sample whencontacted with the biological sample.

In some embodiments, a plurality of molecules (e.g., a plurality ofnucleic acid molecules) having a plurality of barcodes (e.g., aplurality of spatial barcodes) are migrated or transferred from asubstrate to cells of a biological sample. In some embodiments,migrating a plurality of molecules from a substrate to cells of abiological sample includes applying a force (e.g., mechanical,centrifugal, or electrophoretic) to the substrate and/or the biologicalsample to facilitate migration of the plurality of molecules from thesubstrate to the biological sample.

In some embodiments of any of the spatial analysis methods describedherein, physical force is used to facilitate attachment to orintroduction of a molecule (e.g., a nucleic acid molecule) having abarcode (e.g., a spatial barcode) into a biological sample (e.g., a cellpresent in a biological sample). As used herein, “physical force” refersto the use of a physical force to counteract the cell membrane barrierin facilitating intracellular delivery of molecules. Examples ofphysical force instruments and methods that can be used in accordancewith materials and methods described herein include the use of a needle,ballistic DNA, electroporation, sonoporation, photoporation,magnetofection, hydroporation, and combinations thereof.

In some embodiments, biological samples (e.g., cells in a biologicalsample) can be labelled using cell-tagging agents where the cell-taggingagents facilitate the introduction of the molecules (e.g., nucleic acidmolecules) having barcodes (e.g., spatial barcodes) into the biologicalsample (e.g., into cells in a biological sample). As used herein, theterm “cell-tagging agent” refers to a molecule having a moiety that iscapable of attaching to the surface of a cell (e.g., thus attaching thebarcode to the surface of the cell) and/or penetrating and passingthrough the cell membrane (e.g., thus introducing the barcode to theinterior of the cell). In some embodiments, a cell-tagging agentincludes a barcode (e.g., a spatial barcode). The barcode of a barcodedcell-tagging agent can be any of the variety of barcodes describedherein. In some embodiments, the barcode of a barcoded cell-taggingagent is a spatial barcode. In some embodiments, a cell-tagging agentcomprises a nucleic acid molecule that includes the barcode (e.g., thespatial barcode). In some embodiments, the barcode of a barcodedcell-tagging agent identifies the associated molecule, where eachspatial barcode is associated with a particular molecule. In someembodiments, one or more molecules are applied to a sample. In someembodiments, a nucleic acid molecule that includes the barcode iscovalently attached to the cell-tagging agent. In some embodiments, anucleic acid molecule that includes the barcode is non-covalentlyattached to the cell-tagging agent. A non-limiting example ofnon-covalent attachment includes hybridizing the nucleic acid moleculethat includes the barcode to a nucleic acid molecule on the cell-taggingagent (which nucleic acid molecule on the cell-tagging agent can bebound to the cell-tagging agent covalently or non-covalently). In someembodiments, a nucleic acid molecule that is attached to a cell-taggingagent that includes a barcode (e.g., a spatial barcode) also includesone or more additional domains. Such additional domains include, withoutlimitation, a PCR handle, a sequencing priming site, a domain forhybridizing to another nucleic acid molecule, and combinations thereof.

In some embodiments, a cell-tagging agent attaches to the surface of acell. When the cell-tagging agent includes a barcode (e.g., a nucleicacid that includes a spatial barcode), the barcode is also attached tothe surface of the cell. In some embodiments of any of the spatialanalysis methods described herein, a cell-tagging agent attachescovalently to the cell surface to facilitate introduction of the spatialprofiling reagents. In some embodiments of any of the spatial analysismethods described herein, a cell-tagging agent attaches non-covalentlyto the cell surface to facilitate introduction of the spatial profilingreagents.

In some embodiments, once a cell or cells in a biological sample isspatially tagged with a cell-tagging agent(s), spatial analysis ofanalytes present in the biological sample is performed. In someembodiments, such spatial analysis includes dissociating thespatially-tagged cells of the biological sample (or a subset of thespatially-tagged cells of the biological sample) and analyzing analytespresent in those cells on a cell-by-cell basis. Any of a variety ofmethods for analyzing analytes present in cells on a cell-by-cell basiscan be used. Non-limiting examples include any of the variety of methodsdescribed herein and methods described in PCT Application PublicationNo. WO 2019/113533A1, the content of which is incorporated herein byreference in its entirety. For example, the spatially-tagged cells canbe encapsulated with beads comprising one or more nucleic acid moleculeshaving a barcode (e.g., a cellular barcode) (e.g., an emulsion). Thenucleic acid present on the bead can have a domain that hybridizes to adomain on a nucleic acid present on the tagged cell (e.g., a domain on anucleic acid that is attached to a cell-tagging agent), thus linking thespatial barcode of the cell to the cellular barcode of the bead. Oncethe spatial barcode of the cell and the cellular barcode of the bead arelinked, analytes present in the cell can be analyzed using captureprobes (e.g., capture probes present on the bead). This allows thenucleic acids produced (using these methods) from specific cells to beamplified and sequenced separately (e.g. within separate partitions ordroplets).

In some embodiments, once a cell or cells in a biological sample isspatially tagged with a cell-tagging agent(s), spatial analysis ofanalytes present in the biological sample is performed in which thecells of the biological sample are not dissociated into single cells. Insuch embodiments, various methods of spatial analysis such as any ofthose provided herein can be employed. For example, once a cell or cellsin a biological sample is spatially tagged with a cell-tagging agent(s),analytes in the cells can be captured and assayed. In some embodiments,cell-tagging agents include both a spatial barcode and a capture domainthat can be used to capture analytes present in a cell. For example,cell-tagging agents that include both a spatial barcode and a capturedomain can be introduced to cells of the biological sample in a way suchthat locations of the cell-tagging agents are known (or can bedetermined after introducing them to the cells). One non-limitingexample of introducing cell-tagging agents to a biological sample is toprovide the cell-tagging agents in an arrayed format (e.g., arrayed on asubstrate such as any of the variety of substrates and arrays providedherein), where the positions of the cell-tagging agents on the array areknown at the time of introduction (or can be determined afterintroduction). The cells can be permeabilized as necessary (e.g., usingpermeabilization agents and methods described herein), reagents foranalyte analysis can be provided to the cells (e.g., a reversetranscriptase, a polymerase, nucleotides, etc., in the case where theanalyte is a nucleic acid that binds to the capture probe), and theanalytes can be assayed. In some embodiments, the assayed analytes(and/or copies thereof) can be released from the substrate and analyzed.In some embodiments, the assayed analytes (and/or copies thereof) areassayed in situ.

Introducing a Cell-Tagging Agent to the Surface of a Cell

Non-limiting examples of cell-tagging agents and systems that attach tothe surface of a cell (e.g., thus introducing the cell-tagging agent andany barcode attached thereto to the exterior of the cell) that can beused in accordance with materials and methods provided herein forspatially profiling an analyte or analytes in a biological sampleinclude: lipid tagged primers / lipophilic-tagged moieties, positive orneutral oligo-conjugated polymers, antibody-tagged primers,streptavidin-conjugated oligonucleotides, dye-tagged oligonucleotides,click-chemistry, receptor-ligand systems, covalent binding systems viaamine or thiol functionalities, and combinations thereof.

Lipid Tagged Primers / Lipophilic-Tagged Moieties

In some embodiments of any of the spatial profiling methods describedherein, a molecule (e.g., a nucleic acid molecule) having a barcode(e.g., a spatial barcode) is coupled to a lipophilic molecule. In someembodiments, the lipophilic molecule enables the delivery of themolecule to the cell membrane or the nuclear membrane. In someembodiments, a molecule (e.g., a nucleic acid molecule) having a barcode(e.g., a spatial barcode) coupled to a lipophilic molecule can associatewith and/or insert into lipid membranes such as cell membranes andnuclear membranes. In some cases, the insertion can be reversible. Insome cases, the association between the lipophilic molecule and the cellmay be such that the cell retains the lipophilic molecule (e.g., andassociated components, such as nucleic acid barcode molecules) duringsubsequent processing (e.g., partitioning, cell permeabilization,amplification, pooling, etc.). In some embodiments, a molecule (e.g., anucleic acid molecule) having a barcode (e.g., a spatial barcode)coupled to a lipophilic molecule may enter into the intracellular spaceand/or a cell nucleus.

Non-limiting examples of lipophilic molecules that can be used inembodiments described herein include sterol lipids such as cholesterol,tocopherol, steryl, palmitate, lignoceric acid, and derivatives thereof.In some embodiments, the lipophilic molecules are neutral lipids thatare conjugated to hydrophobic moieties (e.g., cholesterol, squalene, orfatty acids) (See Raouane et al. Bioconjugate Chem., 23(6):1091-1104(2012) which is herein incorporated by reference in its entirety). Insome embodiments, a molecule (e.g., a nucleic acid molecule) having abarcode (e.g., a spatial barcode) may be attached to the lipophilicmoiety via a linker, such as a tetra-ethylene glycol (TEG) linker. Otherexemplary linkers include, but are not limited to, Amino Linker C6,Amino Linker C12, Spacer C3, Spacer C6, Spacer C12, Spacer 9, and Spacer18. In some embodiments, a molecule (e.g., a nucleic acid molecule)having a barcode (e.g., a spatial barcode) is indirectly coupled (e.g.,via hybridization or ligand-ligand interactions, such asbiotin-streptavidin) to a lipophilic molecule. Other lipophilicmolecules that may be used in accordance with methods provided hereininclude amphiphilic molecules wherein the headgroup (e.g., charge,aliphatic content, and/or aromatic content) and/or fatty acid chainlength (e.g., C12, C14, C16, or C18) can be varied. For instance, fattyacid side chains (e.g., C12, C14, C16, or C18) can be coupled toglycerol or glycerol derivatives (e.g., 3-t-butyldiphenylsilylglycerol),which can also comprise, e.g., a cationic head group. In someembodiments, a molecule (e.g., a nucleic acid molecule) having a barcode(e.g., a spatial barcode) disclosed herein can then be coupled (eitherdirectly or indirectly) to these amphiphilic molecules. In someembodiments, a molecule (e.g., a nucleic acid molecule) having a barcode(e.g., a spatial barcode) coupled to an amphiphilic molecule mayassociate with and/or insert into a membrane (e.g., a cell, cell bead,or nuclear membrane). In some cases, an amphiphilic or lipophilic moietymay cross a cell membrane and provide a molecule (e.g., a nucleic acidmolecule) having a barcode (e.g., a spatial barcode) to an internalregion of a cell and/or cell bead.

In some embodiments, wherein the molecule (e.g., with a nucleic acidsequence) has an amino group within the molecule, the molecule (e.g., anucleic acid molecule) having a barcode (e.g., a spatial barcode) and anamino group can be coupled to an amine-reactive lipophilic molecule. Forexample, a molecule (e.g., a nucleic acid molecule) having a barcode(e.g., a spatial barcode) and an amino group can be conjugated toDSPE-PEG(2000)-cyanuric chloride(1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[cyanur(polyethyleneglycol)-2000]).

In some embodiments, a cell tagging agent can attach to a surface of acell through a combination of lipophilic and covalent attachment. Forexample, a cell tagging agent can include an oligonucleotide attached toa lipid to target the oligonucleotide to a cell membrane, and an aminegroup that can be covalently linked to a cell surface protein(s) via anynumber of chemistries described herein. In these embodiments, the lipidcan increase the surface concentration of the oligonucleotide and canpromote the covalent reaction.

Positive or Neutral Oligo-Conjugated Polymers

In some embodiments of any of the spatial analysis methods describedherein, a molecule (e.g., a nucleic acid molecule) having a barcode(e.g., a spatial barcode) can be coupled to a glycol chitosanderivative. The glycol chitosan derivative (e.g., glycolchitosan-cholesterol) can serve as a hydrophobic anchor (see Wang et al.J. Mater. Chem. B., 30:6165 (2015), which is herein incorporated byreference in its entirety). Non-limiting examples of chitosanderivatives that can be coupled to a molecule (e.g., a nucleic acidmolecule) having a barcode (e.g., a spatial barcode) can be found inCheung et al., Marine Drugs, 13(8): 5156-5186 (2015), which is hereinincorporated by reference in its entirety.

Antibody-Tagged Primers

In some embodiments of any of the spatial analysis methods describedherein, a molecule (e.g., a nucleic acid molecule) having a barcode(e.g., a spatial barcode) can be coupled to an antibody or antigenbinding fragment thereof in a manner that facilitates attachment of themolecule (e.g., a nucleic acid molecule) having a barcode (e.g., aspatial barcode) to the surface of a cell. In some embodiments,facilitating attachment to the cell surface facilitates introduction ofthe molecule (e.g., a nucleic acid molecule) having a barcode (e.g., aspatial barcode) into the cell. In some embodiments, the molecule (e.g.,a nucleic acid molecule) having a barcode (e.g., a spatial barcode) canbe coupled to an antibody that is directed to an antigen that is presenton the surface of a cell. In some embodiments, the molecule (e.g., anucleic acid molecule) having a barcode (e.g., a spatial barcode) can becoupled to an antibody that is directed to an antigen that is present onthe surface of a plurality of cells (e.g., a plurality of cells in abiological sample). In some embodiments, the molecule (e.g., a nucleicacid molecule) having a barcode (e.g., a spatial barcode) can be coupledto an antibody that is directed to an antigen that is present on thesurface of all or substantially all the cells present in a biologicalsample. Any of the exemplary methods described herein of attaching amolecule (e.g., a nucleic acid molecule) having a barcode (e.g., aspatial barcode) to another molecule (e.g., a cell-tagging agent) can beused.

Streptavidin-Conjugated Oligonucleotides

In some embodiments of any of the spatial analysis methods describedherein, a molecule (e.g., a nucleic acid molecule) having a barcode(e.g., a spatial barcode) can attach to the surface of a cell usingbiotin-streptavidin. In some embodiments, primary amines in the sidechain of lysine residues of cell surface polypeptides are labelled withNHS-activated biotin reagents. For example, the N-terminus of apolypeptide can react with NHS-activated biotin reagents to form stableamide bonds. In some embodiments, cell-tagging agents include molecules(e.g., a nucleic acid molecule) having barcodes (e.g., a spatialbarcode) conjugated to streptavidin. In some cases, streptavidin can beconjugated to the molecule (e.g., a nucleic acid molecule) having abarcode (e.g., a spatial barcode) using click chemistry (e.g., maleimidemodification) as described herein. In some embodiments, a cellcontaining NHS-activated biotin incorporated into lysine side chains ofa cell surface protein forms a non-covalent bond with the streptavidinconjugated to the molecule (e.g., a nucleic acid molecule) having abarcode (e.g., a spatial barcode). In some embodiments, the molecule(e.g., a nucleic acid molecule) having a barcode (e.g., a spatialbarcode) conjugated to streptavidin is itself part of a cell-taggingagent.

Dye-Tagged Oligonucleotides

In some embodiments of any of the spatial analysis methods describedherein, a molecule (e.g., a nucleic acid molecule) having a barcode(e.g., a spatial barcode) is directly linked to a fluorescent tag. Insome embodiments, the physical properties of the fluorescent tags (e.g.,hydrophobic properties) can overcome the hydrophilic nature of themolecule (e.g., a nucleic acid molecule) having a barcode (e.g., aspatial barcode). For example, in some embodiments, wherein the moleculeis a nucleic acid molecule, a fluorescent tag (e.g., BODIPY, Cy3, Atto647N, and Rhodamine Red C2) can be coupled to a 5′ end of the nucleicacid molecule having a barcode (e.g., a spatial barcode). In someembodiments, wherein the molecule is a nucleic acid molecule, anyfluorescent tag having hydrophobic properties can be coupled to thenucleic acid molecule having a barcode (e.g., a spatial barcode) in amanner that overcomes the hydrophilic nature of the nucleic acidmolecule. Non-limiting examples of fluorescent tags with hydrophobicproperties include BODIPY, Cy3, Atto 647N, and Rhodamine Red C2.

Click-Chemistry

In some embodiments of any of the spatial analysis methods describedherein, molecules (e.g., a nucleic acid molecule) having barcodes (e.g.,a spatial barcode) are coupled to click-chemistry moieties. As usedherein, the term “click chemistry,” generally refers to reactions thatare modular, wide in scope, give high yields, generate only inoffensivebyproducts, such as those that can be removed by nonchromatographicmethods, and are stereospecific (but not necessarily enantioselective)(see, e.g., Angew. Chem. Int. Ed., 2001, 40(11):2004-2021, which isincorporated herein by reference in its entirety). In some cases, clickchemistry can describe pairs of functional groups that can selectivelyreact with each other in mild, aqueous conditions.

An example of a click chemistry reaction is the Huisgen 1,3-dipolarcycloaddition of an azide and an alkyne, i.e., copper-catalysed reactionof an azide with an alkyne to form the 5-membered heteroatom ring1,2,3-triazole. The reaction is also known as a Cu(I)-CatalyzedAzide-Alkyne Cycloaddition (CuAAC), a Cu(I) click chemistry or a Cu+click chemistry. Catalysts for the click chemistry include, but are notlimited to, Cu(I) salts, or Cu(I) salts made in situ by reducing Cu(II)reagents to Cu(I) reagents with a reducing reagent (Pharm Res. 2008,25(10): 2216-2230, which is incorporated herein by reference in itsentirety). Known Cu(II) reagents for the click chemistry can include,but are not limited to, the Cu(II)-(TBTA) complex and the Cu(II) (THPTA)complex. TBTA, which istris-[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine, also known astris-(benzyltriazolylmethyl)amine, can be a stabilizing ligand for Cu(I)salts. THPTA, which is tris-(hydroxypropyltriazolylmethyl)amine, isanother example of a stabilizing agent for Cu(I). Other conditions canalso be used to construct the 1,2,3-triazole ring from an azide and analkyne using copper-free click chemistry, such as the Strain-promotedAzide- Alkyne Click chemistry reaction (SPAAC) (see, e.g., Chem.Commun., 2011, 47:6257-6259 and Nature, 2015, 519(7544):486-90, each ofwhich is incorporated herein by reference in its entirety).

Receptor-Ligand Systems

In some embodiments of any of the spatial analysis methods describedherein, a molecule (e.g., a nucleic acid molecule) having a barcode(e.g., a spatial barcode) can be coupled to a ligand, wherein the ligandis part of a receptor-ligand interaction on the surface of a cell. Forexample, a molecule (e.g., a nucleic acid molecule) having a barcode(e.g., a spatial barcode) can be coupled to a ligand that interactsselectively with a cell surface receptor thereby targeting the molecule(e.g., a nucleic acid molecule) having a barcode (e.g., a spatialbarcode) to a specific cell. Non-limiting examples of receptor-ligandsystems that can be used include integrin receptor-ligand interactions,GPCR receptor-ligand interactions, RTK receptor-ligand interactions, andTLR-ligand interactions (see Juliano, Nucleic Acids Res., 44(14):6518-6548 (2016), which is incorporated herein by reference in itsentirety). Any of the methods described herein for attaching a molecule(e.g., a nucleic acid molecule) having a barcode (e.g., a spatialbarcode) to a ligand (e.g., any of the methods described herein relatingto attaching a molecule (e.g., a nucleic acid molecule) having a barcode(e.g., a spatial barcode) to an antibody) can be used.

Covalent Binding Systems via Amine or Thiol Functionalities

In some embodiments of any of the spatial analysis methods describedherein, a molecule (e.g., a nucleic acid molecule) having a barcode(e.g., a spatial barcode) can incorporate reactive functional groups atsites within the molecule (e.g., with a nucleic acid sequence). In suchcases, the reactive functional groups can facilitate conjugation toligands and/or surfaces. In some embodiments, a molecule (e.g., anucleic acid molecule) having a barcode (e.g., a spatial barcode) caninclude thiol modifiers that are designed to react with a broad array ofactivated accepting groups (e.g., maleimide and gold microspheres). Forexample, a molecule (e.g., a nucleic acid molecule) having a barcode(e.g., a spatial barcode) having thiol modifiers can interact with amaleimide-conjugated peptide thereby resulting in labelling of thepeptide. In some embodiments, maleimide-conjugated peptides are presenton the surface of a cell whereupon interaction with the thiol-modifiedmolecule (e.g., a nucleic acid molecule) having a barcode (e.g., aspatial barcode), the molecule (e.g., a nucleic acid molecule) having abarcode (e.g., a spatial barcode) is coupled to the surface of the cell.Non-limiting examples of thiol modifiers include: 5′ thiol modifier C6S-S, 3′ thiol modifier C3 S-S, dithiol, 3′thiol modifier oxa 6-S-S, anddithiol serinol.

In some embodiments of any of the spatial analysis methods describedherein, a molecule (e.g., a nucleic acid molecule) having a barcode(e.g., a spatial barcode) can include amine modifiers, e.g., aminemodifiers that are designed to attach to another molecule in thepresence of an acylating agent. In some embodiments, a molecule (e.g., anucleic acid molecule) having a barcode (e.g., a spatial barcode) caninclude amine modifiers that are designed to attach to a broad array oflinkage groups (e.g., carbonyl amide, thiourea, sulfonamide, andcarboxamide). For example, a molecule (e.g., a nucleic acid molecule)having a barcode (e.g., a spatial barcode) and an amine modifier caninteract with a sulfonamide-conjugated peptide thereby resulting inlabelling of the peptide. In some embodiments, sulfonamide-conjugatedpeptides are present on the surface of a cell whereupon interaction withthe amine-modified molecule (e.g., a nucleic acid molecule) having abarcode (e.g., a spatial barcode), the molecule (e.g., a nucleic acidmolecule) having a barcode (e.g., a spatial barcode) is coupled to thesurface of the cell. Non-limiting example of amine modifiers include:DMS(O)MT-Amino-Modifier-C6, Amino-Modifier-C3-TFA, Amino-Modifier-C12,Amino-Modifier-C6-TFA, Amino-dT, Amino-Modifier-5, Amino-Modifier-C2-dT,Amino-Modifier-C6-dT, and 3′-Amino-Modifier-C7.

As another example, a molecule (e.g., a nucleic acid molecule) having abarcode (e.g., a spatial barcode) can incorporate reactive functionalgroups at sites within the molecule (e.g., with a nucleic acid sequence)such as N-hydroxysuccinimide (NHS). In some embodiments, amines (e.g.,amine-containing peptides) are present on the surface of a cellwhereupon interaction with the NHS-modified molecule (e.g., a nucleicacid molecule) having a barcode (e.g., a spatial barcode), the molecule(e.g., a nucleic acid molecule) having a barcode (e.g., a spatialbarcode) is coupled to the surface of the cell. In some embodiments, amolecule (e.g., a nucleic acid molecule) having a barcode (e.g., aspatial barcode) is reacted with a bifunctional NHS linker to form anNHS-modified molecule (e.g., a nucleic acid molecule) having a barcode(e.g., a spatial barcode).

In some embodiments, a molecule (e.g., a nucleic acid molecule) having abarcode (e.g., a spatial barcode) can be coupled to a biocompatibleanchor for cell membrane (BAM). For example, a BAM can include moleculesthat comprise an oleyl group and PEG. The oleyl group can facilitateanchoring the molecule (e.g., a nucleic acid molecule) having a barcode(e.g., a spatial barcode) to a cell, and the PEG can increase watersolubility. In some embodiments, oleyl-PEG-NHS can be coupled to amolecule (e.g., a nucleic acid molecule) having a barcode (e.g., aspatial barcode) using NHS chemistry.

Azide-Based Systems

In some embodiments, wherein the molecule (e.g., with a nucleic acidsequence) incorporates reactive functional groups at sites within themolecule, a molecule (e.g., a nucleic acid molecule) having a barcode(e.g., a spatial barcode) can be coupled to an azide group on a cellsurface. In some embodiments, the reactive functional group is analkynyl group. In some embodiments, click chemistry as described hereincan be used to attach the alkynyl-modified molecule (e.g., a nucleicacid molecule) having a barcode (e.g., a spatial barcode) to an azidegroup on the cell surface. An azide group can be attached to the cellsurface through a variety of methods. For example, NHS chemistry can beused to attach an azide group to the cell surface. In some embodiments,N-azidoacetylmannosamine-tetraacylated (Ac4ManNAz), which contains anazide group, can react with sialic acid on the surface of a cell toattach azide to the cell surface. In some embodiments, azide is attachedto the cell surface by bio-orthogonal expression of azide.

Lectin-Based Systems

In some embodiments of any of the spatial analysis methods describedherein, a molecule (e.g., a nucleic acid molecule) having a barcode(e.g., a spatial barcode) can be coupled to a lectin that facilitatesattachment of the molecule (e.g., a nucleic acid molecule) having abarcode (e.g., a spatial barcode) to a cell surface. Lectin can bind toglycans, e.g., glycans on the surface of cells. In some embodiments, themolecule (e.g., a nucleic acid molecule) having a barcode (e.g., aspatial barcode) has an incorporated reactive functional group such asan azide group. In some embodiments, the molecule (e.g., a nucleic acidmolecule) having a barcode (e.g., a spatial barcode) and an azide groupis reacted with a modified lectin, e.g., a lectin modified using NHSchemistry to introduce an azide reactive group. In some embodiments, alive cell is labelled with a lectin-modified molecule (e.g., a nucleicacid molecule) having a barcode (e.g., a spatial barcode). In someembodiments, a fixed cell is labelled with a lectin-modified molecule(e.g., a nucleic acid molecule) having a barcode (e.g., a spatialbarcode). In some embodiments, a permeabilized cell is labelled with alectin-modified molecule (e.g., a nucleic acid molecule) having abarcode (e.g., a spatial barcode). In some embodiments, organelles inthe secretory pathway can be labelled with a lectin-modified molecule(e.g., a nucleic acid molecule) having a barcode (e.g., a spatialbarcode).

(B) Introducing a Cell-Tagging Agent to the Interior of a Cell

Non-limiting examples of cell-tagging agents and systems that penetrateand/or pass through the cell membrane (e.g., thus introducing thecell-tagging agent and any barcode attached thereto to the interior ofthe cell) that can be used in accordance with materials and methodsprovided herein for spatially profiling an analyte or analytes in abiological sample include: a cell-penetrating agent (e.g., acell-penetrating peptide), a nanoparticle, a liposome, a polymersome, apeptide-based chemical vector, electroporation, sonoporation, lentiviralvectors, retroviral vectors, and combinations thereof.

FIG. 19 is a schematic showing an exemplary cell tagging method.Non-exhaustive examples of oligo delivery vehicles may include a cellpenetrating peptide or a nanoparticle. Non-exhaustive examples of thedelivery systems can include lipid-based polymeric and metallicnanoparticles or oligos that can be conjugated or encapsulated withinthe delivery system. The cell tag can be used in combination with acapture agent barcode domain and a cleavable or non-cleavable spatiallybarcoded capture probes for spatial and multiplexing applications.

Cell-Penetrating Agent

In some embodiments of any of the spatial profiling methods describedherein, capture of a biological analyte by a molecule (e.g., a nucleicacid molecule) having a barcode (e.g., a spatial barcode) and a capturedomain is facilitated by a cell-penetrating agent. In some embodiments,a molecule (e.g., a nucleic acid molecule) having a barcode (e.g., aspatial barcode) and a capture domain is coupled to a cell-penetratingagent, and the cell-penetrating agent allows the molecule to interactwith an analyte inside the cell. A “cell-penetrating agent” as usedherein refers to an agent capable of facilitating the introduction of amolecule (e.g., a nucleic acid molecule) having a barcode (e.g., aspatial barcode) and a capture domain into a cell of a biological sample(see, e.g., Lovatt et al. Nat Methods. 2014 Feb;11(2):190-6, which isincorporated herein by reference in its entirety). In some embodiments,a cell-penetrating agent is a cell-penetrating peptide. A“cell-penetrating peptide” as used herein refers to a peptide (e.g., ashort peptide, e.g., a peptide not usually exceeding 30 residues) thathas the capacity to cross cellular membranes.

In some embodiments of any of the spatial profiling methods describedherein, a cell-penetrating peptide coupled to a molecule (e.g., anucleic acid molecule) having a barcode (e.g., a spatial barcode) and acapture domain can cross a cellular membrane using an energy dependentor an energy independent mechanism. For example, a cell-penetratingpeptide can cross a cellular membrane through direct translocationthrough physical perturbation of the plasma membrane, endocytosis,adaptive translocation, pore-formation, electroporation-likepermeabilization, and/or entry at microdomain boundaries. Non-limitingexamples of a cell-penetrating peptide include: penetratin, tat peptide,pVEC, transportan, MPG, Pep-1, a polyarginine peptide, MAP, R6W3,(D-Arg)9, Cys(Npys)-(D-Arg)9, Anti-BetaGamma (MPS -Phosducin - likeprotein C terminus), Cys(Npys) antennapedia, Cys(Npys)-(Arg)9,Cys(Npys)-TAT (47-57), HIV-1 Tat (48-60), KALA, mastoparan,penetratin-Arg, pep-1-cysteamine, TAT(47-57)GGG-Cys(Npys), Tat-NR2Bct,transdermal peptide, SynB1, SynB3, PTD-4, PTD-5, FHV Coat-(35-49), BMVGag-(7-25), HTLV-II Rex-(4-16), R9-tat, SBP, FBP, MPG, MPG(ΔNLS), Pep-2,MTS, plsl, and a polylysine peptide (see, e.g., Bechara et al. FEBSLett. 2013 Jun 19;587(12):1693-702, which is incorporated by referenceherein in its entirety).

Nanoparticles

In some embodiments of any of the spatial profiling methods describedherein, capture of a biological analyte by a molecule (e.g., a nucleicacid molecule) having a barcode (e.g., a spatial barcode) and a capturedomain is facilitated by an inorganic particle (e.g., a nanoparticle).In some embodiments, a molecule (e.g., a nucleic acid molecule) having abarcode (e.g., a spatial barcode) and a capture domain is coupled to aninorganic particle (e.g., a nanoparticle), and the molecule (e.g., anucleic acid molecule) having a barcode (e.g., a spatial barcode) and acapture domain uses the nanoparticle to get access to analytes insidethe cell. Non-limiting examples of nanoparticles that can be used inembodiments herein to deliver a molecule (e.g., a nucleic acid molecule)having a barcode (e.g., a spatial barcode) and a capture domain into acell and/or cell bead include inorganic nanoparticles prepared frommetals, (e.g., iron, gold, and silver), inorganic salts, and ceramics(e.g., phosphate or carbonate salts of calcium, magnesium, or silicon).The surface of a nanoparticle can be coated to facilitate binding of themolecule (e.g., a nucleic acid molecule) having a barcode (e.g., aspatial barcode) and a capture domain, or the surface can be chemicallymodified to facilitate attachment of the molecule (e.g., a nucleic acidmolecule) having a barcode (e.g., a spatial barcode) and a capturedomain. Magnetic nanoparticles (e.g., supermagnetic iron oxide),fullerenes (e.g., soluble carbon molecules), carbon nanotubes (e.g.,cylindrical fullerenes), quantum dots and supramolecular systems canalso be used.

Liposomes

In some embodiments of any of the spatial analysis methods describedherein, capture of a biological analyte by a molecule (e.g., a nucleicacid molecule) having a barcode (e.g., a spatial barcode) and a capturedomain is facilitated by a liposome. Various types of lipids, includingcationic lipids, can be used in liposome delivery. In some cases, amolecule (e.g., a nucleic acid molecule) having a barcode (e.g., aspatial barcode) and a capture domain is delivered to a cell via a lipidnano-emulsion. A lipid emulsion refers to a dispersion of one immiscibleliquid in another stabilized by emulsifying agent. Labeling cells cancomprise use of a solid lipid nanoparticle.

Polymersomes

In some embodiments of any of the spatial analysis methods describedherein, capture of a biological analyte by a molecule (e.g., a nucleicacid molecule) having a barcode (e.g., a spatial barcode) and a capturedomain is facilitated by a polymersome. In some embodiments, a molecule(e.g., a nucleic acid molecule) having a barcode (e.g., a spatialbarcode) and a capture domain is contained in the polymersome, and themolecule (e.g., a nucleic acid molecule) having a barcode (e.g., aspatial barcode) and a capture domain uses the polymersome to get accessto analytes inside the cell. A “polymersome” as referred to herein is anartificial vesicle. For example, a polymersome can be a vesicle similarto a liposome, but the membrane comprises amphiphilic synthetic blockcopolymers (see, e.g., Rideau et al. Chem. Soc. Rev., 2018, 47,8572-8610, which is incorporated by reference herein in its entirety).In some embodiments, polymersomes comprise di-(AB) or tri-blockcopolymers (e.g., ABA or ABC), where A and C are a hydrophilic block andB is a hydrophobic block. In some embodiments, a polymersome comprisespoly(butadiene)-b-poly(ethylene oxide), poly(ethylethylene)-b-poly(ethylene oxide), polystyrene-b-poly(ethylene oxide),poly(2-vinylpyridine)-b- poly(ethylene oxide),polydimethylsiloxane-b-poly(ethylene oxide), polydimethylsiloxane-g-poly(ethylene oxide), polycaprolactone-b-poly(ethylene oxide),polyisobutylene-b-poly(ethylene oxide), polystyrene-b- polyacrylic acid,polydimethylsiloxane-b- poly-2-methyl-2-oxazoline, or a combinationthereof (wherein b=block and g=grafted).

Peptide-Based Chemical Vectors

In some embodiments of any of the spatial analysis methods describedherein, capture of a biological analyte by a molecule (e.g., a nucleicacid molecule) having a barcode (e.g., a spatial barcode) and a capturedomain is facilitated by a peptide-based chemical vector, e.g., acationic peptide-based chemical vector. Cationic peptides can be rich inbasic residues like lysine and/or arginine. In some embodiments of anyof the spatial analysis methods described herein, capture of abiological analyte by a molecule (e.g., a nucleic acid molecule) havinga barcode (e.g., a spatial barcode) and a capture domain is facilitatedby a polymer-based chemical vector. Cationic polymers, when mixed with amolecule (e.g., a nucleic acid molecule) having a barcode (e.g., aspatial barcode) and a capture domain, can form nanosized complexescalled polyplexes. Polymer based vectors can comprise natural proteins,peptides and/or polysaccharides. Polymer based vectors can comprisesynthetic polymers. In some embodiments, a polymer-based vectorcomprises polyethylenimine (PEI). PEI can condense DNA intopositively-charged particles, which bind to anionic cell surfaceresidues and are brought into the cell via endocytosis. In someembodiments, a polymer-based chemical vector comprises poly(L)-lysine(PLL), poly (DL-lactic acid) (PLA), poly (DL-lactide-co-glycoside)(PLGA), polyornithine, polyarginine, histones, protamines, or acombination thereof. Polymer-based vectors can comprise a mixture ofpolymers, for example, PEG and PLL. Other non-limiting examples ofpolymers include dendrimers, chitosans, synthetic amino derivatives ofdextran, and cationic acrylic polymers.

Electroporation

In some embodiments of any of the spatial analysis methods describedherein, capture of a biological analyte by a molecule (e.g., a nucleicacid molecule) having a barcode (e.g., a spatial barcode) and a capturedomain is facilitated by electroporation. With electroporation, abiological analyte by a molecule (e.g., a nucleic acid molecule) havinga barcode (e.g., a spatial barcode) and a capture domain can enter acell through one or more pores in the cellular membrane formed byapplied electricity. The pore of the membrane can be reversible based onthe applied field strength and pulse duration.

Sonoporation

In some embodiments of any of the spatial analysis methods describedherein, capture of a biological analyte by a molecule (e.g., a nucleicacid molecule) having a barcode (e.g., a spatial barcode) and a capturedomain is facilitated by sonoporation. Cell membranes can be temporarilypermeabilized using sound waves, allowing cellular uptake of abiological analyte by a molecule (e.g., a nucleic acid molecule) havinga barcode (e.g., a spatial barcode) and a capture domain.

Lentiviral Vectors and Retroviral Vectors

In some embodiments of any of the spatial analysis methods describedherein, capture of a biological analyte by a molecule (e.g., a nucleicacid molecule) having a barcode (e.g., a spatial barcode) and a capturedomain is facilitated by vectors. For example, a vector as describedherein can be an expression vector where the expression vector includesa promoter sequence operably linked to the sequence encoding themolecule (e.g., a nucleic acid molecule) having a barcode (e.g., aspatial barcode) and a capture domain. Non-limiting examples of vectorsinclude plasmids, transposons, cosmids, and viral vectors (e.g., anyadenoviral vectors (e.g., pSV or pCMV vectors), adeno-associated virus(AAV) vectors, lentivirus vectors, and retroviral vectors), and anyGateway® vectors. A vector can, for example, include sufficientcis-acting elements for expression where other elements for expressioncan be supplied by the host mammalian cell or in an in vitro expressionsystem. Skilled practitioners will be capable of selecting suitablevectors and mammalian cells for introducing any of spatial profilingreagents described herein.

Other Methods and Cell-Tagging Agents for Intracellular Introduction ofa Molecule

In some embodiments of any of the spatial analysis methods describedherein, capture of a biological analyte by a molecule (e.g., a nucleicacid molecule) having a barcode (e.g., a spatial barcode) and a capturedomain is facilitated by the use of a needle, for example for injection(e.g., microinjection), particle bombardment, photoporation,magnetofection, and/or hydroporation. For example, with particlebombardment, a molecule (e.g., a nucleic acid molecule) having a barcode(e.g., a spatial barcode) and a capture domain can be coated with heavymetal particles and delivered to a cell at a high speed. Inphotoporation, a transient pore in a cell membrane can be generatedusing a laser pulse, allowing cellular uptake of a molecule (e.g., anucleic acid molecule) having a barcode (e.g., a spatial barcode) and acapture domain. In magnetofection, a molecule (e.g., a nucleic acidmolecule) having a barcode (e.g., a spatial barcode) and a capturedomain can be coupled to a magnetic particle (e.g., magneticnanoparticle, nanowires, etc.) and localized to a target cell via anapplied magnetic field. In hydroporation, a molecule (e.g., a nucleicacid molecule) having a barcode (e.g., a spatial barcode) and a capturedomain can be delivered to a cell and/or cell bead via hydrodynamicpressure.

(C) Methods for Separating Sample Into Single Cells or Cell Groups

Some embodiments of any of the methods described herein can includeseparating a biological sample into single cells, cell groups, types ofcells, or a region or regions of interest. For example, a biologicalsample can be separated into single cells, cell groups, types of cells,or a region or regions of interest before being contacted with one ormore capture probes. In other examples, a biological sample is firstcontacted with one or more capture probes, and then separated intosingle cells, cell groups, types of cells, or a region or regions ofinterest.

In some embodiments, a biological sample can be separated into chucksusing pixelation. Pixelation can include the steps of providing abiological sample, and punching out one or more portions of thebiological sample. The punched out portions of the biological sample canthen be used to perform any of the methods described herein. In someembodiments, the punched-out portions of the biological sample can be ina random pattern or a designed pattern. In some embodiments, thepunched-out portions of the biological sample can be focused on a regionof interest or a subcellular structure in the biological sample.

FIG. 20A is a workflow schematic illustrating exemplary, non-limiting,non-exhaustive steps for “pixelating” a sample, wherein the sample iscut, stamped, microdissected, or transferred by hollow-needle ormicroneedle, moving a small portion of the sample into an individualpartition or well.

FIG. 20B is a schematic depicting multi-needle pixelation, wherein anarray of needles punched through a sample on a scaffold and intonanowells containing gel beads and reagents below. Once the needle is inthe nanowell, the cell(s) are ejected.

In some embodiments, a biological sample is divided into chucks beforeperformance of any of the spatial analysis methods described herein. Insome embodiments, the methods can include spatial barcoding of FFPE“chunks” via barcodes applied in spatially well-defined pattern (like inDNA microarray printing). The DNA barcode is either long so that it willnot diffuse out in subsequent steps or is covalently applied to the FFPEsample. To enable barcodes to get embedded into an FFPE slide, the waxcan be heated, barcodes can be added to the slide before cooling, andthen the chunks can be cut. The cutting can be done in various ways suchas using laser microdissection, or via mechanical or acoustic means.Other alternates are to embed some fluorophores/Qdots, etc. to preservespatial information into the sample. The barcoding at this step enablesmassively parallel random encapsulation of chunks while retaining localspatial information (e.g., tumor vs normal cells).

In some embodiments, a biological sample can be divided or portionedusing laser capture microdissection (e.g., highly-multiplexed lasercapture microdissection).

(D) Release and Amplification of Analytes

In some embodiments, lysis reagents can be added to the sample tofacilitate the release of analyte(s) from a sample. Examples of lysisagents include, but are not limited to, bioactive reagents such as lysisenzymes that are used for lysis of different cell types, e.g., grampositive or negative bacteria, plants, yeast, mammalian, such aslysozymes, achromopeptidase, lysostaphin, labiase, kitalase, lyticase,and a variety of other commercially available lysis enzymes. Other lysisagents can additionally or alternatively be co-partitioned with thebiological sample to cause the release of the sample’s contents into thepartitions. In some embodiments, surfactant-based lysis solutions can beused to lyse cells, although these can be less desirable foremulsion-based systems where the surfactants can interfere with stableemulsions. Lysis solutions can include ionic surfactants such as, forexample, sarcosyl and sodium dodecyl sulfate (SDS). Electroporation,thermal, acoustic or mechanical cellular disruption can also be used incertain embodiments, e.g., non-emulsion based partitioning such asencapsulation of biological materials that can be in addition to or inplace of droplet partitioning, where any pore size of the encapsulate issufficiently small to retain nucleic acid fragments of a given size,following cellular disruption.

In addition to the permeabilization agents, other reagents can also beadded to interact with the biological sample, including, for example,DNase and RNase inactivating agents or inhibitors, such as proteinase K,chelating agents, such as EDTA, and other reagents to allow forsubsequent processing of analytes from the sample.

Further reagents that can be added to a sample, include, for example,endonucleases to fragment DNA, DNA polymerase enzymes, and dNTPs used toamplify nucleic acids. Other enzymes that can also be added to thesample include, but are not limited to, polymerase, transposase, ligase,proteinase K, and DNAse, etc. Additional reagents can also includereverse transcriptase enzymes, including enzymes with terminaltransferase activity, primers, and switch oligonucleotides. In someembodiments, template switching can be used to increase the length of acDNA, e.g., by appending a predefined nucleic acid sequence to the cDNA.

If a tissue sample is not permeabilized sufficiently, the amount ofanalyte captured on the substrate can be too low to enable adequateanalysis. Conversely, if the tissue sample is too permeable, the analytecan diffuse away from its origin in the tissue sample, such that therelative spatial relationship of the analytes within the tissue sampleis lost. Hence, a balance between permeabilizing the tissue sampleenough to obtain good signal intensity while still maintaining thespatial resolution of the analyte distribution in the tissue sample isdesired.

In some embodiments, where the biological sample includes live cells,permeabilization conditions can be modified so that the live cellsexperience only brief permeabilization (e.g., through short repetitivebursts of electric field application), thereby allowing one or moreanalytes to migrate from the live cells to the substrate while retainingcellular viability.

In some embodiments, after contacting a biological sample with asubstrate that include capture probes, a removal step is performed toremove all or a portion of the biological sample from the substrate. Insome embodiments, the removal step includes enzymatic or chemicaldegradation of the permeabilized cells of the biological sample. Forexample, the removal step can include treating the biological sampleswith an enzyme (e.g., proteinase K) to remove at least a portion of thebiological sample from the first substrates. In some embodiments, theremoval step can include ablation of the tissue (e.g., laser ablation).

In some embodiments, where RNA is captured from cells in a sample, oneor more RNA species of interest can be selectively enriched. Forexample, one or more species of RNA of interest can be selected byaddition of one or more oligonucleotides. One or more species of RNA canbe selectively down-selected (e.g., removed) using any of a variety ofmethods. For example, probes can be administered to a sample thatselectively hybridize to ribosomal RNA (rRNA), thereby reducing the pooland concentration of rRNA in the sample. Subsequent application of thecapture probes to the sample can result in improved RNA capture due tothe reduction in non-specific RNA present in the sample. In someembodiments, the additional oligonucleotide is a sequence used forpriming a reaction by a polymerase. For example, one or more primersequences with sequence complementarity to one or more RNAs of interest,can be used to amplify the one or more RNAs of interest, therebyselectively enriching these RNAs. In some embodiments, anoligonucleotide with sequence complementarity to the complementarystrand of captured RNA (e.g., cDNA) can bind to the cDNA. In onenon-limiting example, biotinylated oligonucleotides with sequencecomplementary to one or more cDNA of interest binds to the cDNA and canbe selected using biotinylation-strepavidin affinity in any number ofmethods known to the field (e.g., streptavidin beads).

Nucleic acid analytes can be amplified using a polymerase chain reaction(e.g., digital PCR, quantitative PCR, or real time PCR), or isothermalamplification, or any of the nucleic acid amplification or extensionreactions described herein.

(E) Partitioning

As discussed above, in some embodiments, the sample can optionally beseparated into single cells, cell groups, or other fragments/pieces thatare smaller than the original, unfragmented sample. Each of thesesmaller portions of the sample can be analyzed to obtainspatially-resolved analyte information from the sample. Non-limitingpartitioning methods are described herein.

For samples that have been separated into smaller fragments - andparticularly, for samples that have been disaggregated, dissociated, orotherwise separated into individual cells -one method for analyzing thefragments involves partitioning the fragments into individual partitions(e.g., fluid droplets), and then analyzing the contents of thepartitions. In general, each partition maintains separation of its owncontents from the contents of other partitions. For example, thepartition can be a droplet in an emulsion.

In addition to analytes, a partition can include additional components,and in particular, one or more beads. A partition can include a singlegel bead, a single cell bead, or both a single cell bead and single gelbead.

A partition can also include one or more reagents. Unique identifiers,such as barcodes, can be injected into the droplets previous to,subsequent to, or concurrently with droplet generation, such as via amicrocapsule (e.g., bead). Microfluidic channel networks (e.g., on achip) can be utilized to generate partitions. Alternative mechanisms canalso be employed in the partitioning of individual biological particles,including porous membranes through which aqueous mixtures of cells areextruded into non-aqueous fluids.

The partitions can be flowable within fluid streams. The partitions caninclude, for example, micro-vesicles that have an outer barriersurrounding an inner fluid center or core. In some cases, the partitionscan include a porous matrix that is capable of entraining and/orretaining materials within its matrix. The partitions can be droplets ofa first phase within a second phase, wherein the first and second phasesare immiscible. For example, the partitions can be droplets of aqueousfluid within a non-aqueous continuous phase (e.g., oil phase). Inanother example, the partitions can be droplets of a non-aqueous fluidwithin an aqueous phase. In some examples, the partitions can beprovided in a water-in-oil emulsion or oil-in-water emulsion. A varietyof different vessels are described in, for example, U.S. Pat.Application Publication No. 2014/0155295, the entire contents of whichare incorporated herein by reference. Emulsion systems for creatingstable droplets in non-aqueous or oil continuous phases are described,for example, in U.S. Pat. Application Publication No. 2010/0105112, theentire contents of which are incorporated herein by reference.

For droplets in an emulsion, allocating individual particles to discretepartitions can be accomplished, for example, by introducing a flowingstream of particles in an aqueous fluid into a flowing stream of anon-aqueous fluid, such that droplets are generated at the junction ofthe two streams. Fluid properties (e.g., fluid flow rates, fluidviscosities, etc.), particle properties (e.g., volume fraction, particlesize, particle concentration, etc.), microfluidic architectures (e.g.,channel geometry, etc.), and other parameters can be adjusted to controlthe occupancy of the resulting partitions (e.g., number of analytes perpartition, number of beads per partition, etc.) For example, partitionoccupancy can be controlled by providing the aqueous stream at a certainconcentration and/or flow rate of analytes.

To generate single analyte partitions, the relative flow rates of theimmiscible fluids can be selected such that, on average, the partitionscan contain less than one analyte per partition to ensure that thosepartitions that are occupied are primarily singly occupied. In somecases, partitions among a plurality of partitions can contain at mostone analyte. In some embodiments, the various parameters (e.g., fluidproperties, particle properties, microfluidic architectures, etc.) canbe selected or adjusted such that a majority of partitions are occupied,for example, allowing for only a small percentage of unoccupiedpartitions. The flows and channel architectures can be controlled as toensure a given number of singly occupied partitions, less than a certainlevel of unoccupied partitions and/or less than a certain level ofmultiply occupied partitions.

The channel segments described herein can be coupled to any of a varietyof different fluid sources or receiving components, includingreservoirs, tubing, manifolds, or fluidic components of other systems.As will be appreciated, the microfluidic channel structure can have avariety of geometries. For example, a microfluidic channel structure canhave one or more than one channel junction. As another example, amicrofluidic channel structure can have 2, 3, 4, or 5 channel segmentseach carrying particles that meet at a channel junction. Fluid can bedirected to flow along one or more channels or reservoirs via one ormore fluid flow units. A fluid flow unit can include compressors (e.g.,providing positive pressure), pumps (e.g., providing negative pressure),actuators, and the like to control flow of the fluid. Fluid can also orotherwise be controlled via applied pressure differentials, centrifugalforce, electrokinetic pumping, vacuum, capillary, and/or gravity flow.

A partition can include one or more unique identifiers, such asbarcodes. Barcodes can be previously, subsequently, or concurrentlydelivered to the partitions that hold the compartmentalized orpartitioned biological particle. For example, barcodes can be injectedinto droplets previous to, subsequent to, or concurrently with dropletgeneration. The delivery of the barcodes to a particular partitionallows for the later attribution of the characteristics of theindividual biological particle to the particular partition. Barcodes canbe delivered, for example on a nucleic acid molecule (e.g., anoligonucleotide), to a partition via any suitable mechanism. Barcodednucleic acid molecules can be delivered to a partition via amicrocapsule. A microcapsule, in some instances, can include a bead.

In some embodiments, barcoded nucleic acid molecules can be initiallyassociated with the microcapsule and then released from themicrocapsule. Release of the barcoded nucleic acid molecules can bepassive (e.g., by diffusion out of the microcapsule). In addition oralternatively, release from the microcapsule can be upon application ofa stimulus which allows the barcoded nucleic acid nucleic acid moleculesto dissociate or to be released from the microcapsule. Such stimulus candisrupt the microcapsule, an interaction that couples the barcodednucleic acid molecules to or within the microcapsule, or both. Suchstimulus can include, for example, a thermal stimulus, photo-stimulus,chemical stimulus (e.g., change in pH or use of a reducing agent(s)), amechanical stimulus, a radiation stimulus; a biological stimulus (e.g.,enzyme), or any combination thereof.

In some embodiments, one more barcodes (e.g., spatial barcodes, UMIs, ora combination thereof) can be introduced into a partition as part of theanalyte. As described previously, barcodes can be bound to the analytedirectly, or can form part of a capture probe or analyte capture agentthat is hybridized to, conjugated to, or otherwise associated with ananalyte, such that when the analyte is introduced into the partition,the barcode(s) are introduced as well.

FIG. 21 depicts an exemplary workflow, where a sample is contacted witha spatially-barcoded capture probe array and the sample is fixed,stained, and imaged 2101, as described elsewhere herein. The captureprobes can be cleaved from the array 2102 using any method as describedherein. The capture probes can diffuse toward the cells by eitherpassive or active migration as described elsewhere herein. The captureprobes may then be introduced to the sample 2103 as described elsewhereherein, wherein the capture probe is able to gain entry into the cell inthe absence of cell permeabilization, using one of the cell penetratingpeptides or lipid delivery systems described herein. The sample can thenbe optionally imaged in order to confirm probe uptake, via a reportermolecule incorporated within the capture probe 2104. The sample can thenbe separated from the array and undergo dissociation 2105, wherein thesample is separated into single cells or small groups of cells. Once thesample is dissociated, the single cells can be introduced to an oil-inwater droplet 2106, wherein a single cell is combined with reagentswithin the droplet and processed so that the spatial barcode thatpenetrated the cell labels the contents of that cell within the droplet.Other cells undergo separately partitioned reactions concurrently. Thecontents of the droplet is then sequenced 2107 in order to associate aparticular cell or cells with a particular spatial location within thesample 2108.

As described above, FIG. 16 shows an example of a microfluidic channelstructure for partitioning individual analytes (e.g., cells) intodiscrete partitions. FIGS. 17A and 17C also show other examples ofmicrofluidic channel structures that can be used for delivering beads todroplets.

A variety of different beads can be incorporated into partitions asdescribed above. In some embodiments, for example, non-barcoded beadscan be incorporated into the partitions. For example, where thebiological particle (e.g., a cell) that is incorporated into thepartitions carries one or more barcodes (e.g., spatial barcode(s),UMI(s), and combinations thereof), the bead can be a non-barcoded bead.

In some embodiments, a barcode carrying bead can be incorporated intopartitions. For example, a nucleic acid molecule, such as anoligonucleotide, can be coupled to a bead by a releasable linkage, suchas, for example, a disulfide linker. The same bead can be coupled (e.g.,via releasable linkage) to one or more other nucleic acid molecules. Thenucleic acid molecule can be or include a barcode. As noted elsewhereherein, the structure of the barcode can include a number of sequenceelements.

The nucleic acid molecule can include a functional domain that can beused in subsequent processing. For example, the functional domain caninclude one or more of a sequencer specific flow cell attachmentsequence (e.g., a P5 sequence for Illumina® sequencing systems) and asequencing primer sequence (e.g., a R1 primer for Illumina® sequencingsystems). The nucleic acid molecule can include a barcode sequence foruse in barcoding the sample (e.g., DNA, RNA, protein, etc.). In somecases, the barcode sequence can be bead-specific such that the barcodesequence is common to all nucleic acid molecules coupled to the samebead. Alternatively or in addition, the barcode sequence can bepartition-specific such that the barcode sequence is common to allnucleic acid molecules coupled to one or more beads that are partitionedinto the same partition. The nucleic acid molecule can include aspecific priming sequence, such as an mRNA specific priming sequence(e.g., poly (T) sequence), a targeted priming sequence, and/or a randompriming sequence. The nucleic acid molecule can include an anchoringsequence to ensure that the specific priming sequence hybridizes at thesequence end (e.g., of the mRNA). For example, the anchoring sequencecan include a random short sequence of nucleotides, such as a 1-mer,2-mer, 3-mer or longer sequence, which can ensure that a poly(T) segmentis more likely to hybridize at the sequence end of the poly(A) tail ofthe mRNA.

The nucleic acid molecule can include a unique molecular identifyingsequence (e.g., unique molecular identifier (UMI)). In some embodiments,the unique molecular identifying sequence can include from about 5 toabout 8 nucleotides. Alternatively, the unique molecular identifyingsequence can include less than about 5 or more than about 8 nucleotides.The unique molecular identifying sequence can be a unique sequence thatvaries across individual nucleic acid molecules coupled to a singlebead.

In some embodiments, the unique molecular identifying sequence can be arandom sequence (e.g., such as a random N-mer sequence). For example,the UMI can provide a unique identifier of the starting mRNA moleculethat was captured, in order to allow quantitation of the number oforiginal expressed RNA.

In general, an individual bead can be coupled to any number ofindividual nucleic acid molecules, for example, from one to tens tohundreds of thousands or even millions of individual nucleic acidmolecules. The respective barcodes for the individual nucleic acidmolecules can include both common sequence segments or relatively commonsequence segments and variable or unique sequence segments betweendifferent individual nucleic acid molecules coupled to the same bead.

Within any given partition, all of the cDNA transcripts of theindividual mRNA molecules can include a common barcode sequence segment.However, the transcripts made from the different mRNA molecules within agiven partition can vary at the unique molecular identifying sequencesegment (e.g., UMI segment). Beneficially, even following any subsequentamplification of the contents of a given partition, the number ofdifferent UMIs can be indicative of the quantity of mRNA originatingfrom a given partition. As noted above, the transcripts can beamplified, cleaned up and sequenced to identify the sequence of the cDNAtranscript of the mRNA, as well as to sequence the barcode segment andthe UMI segment. While a poly(T) primer sequence is described, othertargeted or random priming sequences can also be used in priming thereverse transcription reaction. Likewise, although described asreleasing the barcoded oligonucleotides into the partition, in somecases, the nucleic acid molecules bound to the bead can be used tohybridize and capture the mRNA on the solid phase of the bead, forexample, in order to facilitate the separation of the RNA from othercell contents.

In some embodiments, precursors that include a functional group that isreactive or capable of being activated such that it becomes reactive canbe polymerized with other precursors to generate gel beads that includethe activated or activatable functional group. The functional group canthen be used to attach additional species (e.g., disulfide linkers,primers, other oligonucleotides, etc.) to the gel beads. For example,some precursors featuring a carboxylic acid (COOH) group canco-polymerize with other precursors to form a bead that also includes aCOOH functional group. In some cases, acrylic acid (a species comprisingfree COOH groups), acrylamide, and bis(acryloyl)cystamine can beco-polymerized together to generate a bead with free COOH groups. TheCOOH groups of the bead can be activated (e.g., via1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) andN-Hydroxysuccinimide (NHS) or4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride(DMTMM)) such that they are reactive (e.g., reactive to amine functionalgroups where EDC/NHS or DMTMM are used for activation). The activatedCOOH groups can then react with an appropriate species (e.g., a speciescomprising an amine functional group where the carboxylic acid groupsare activated to be reactive with an amine functional group) comprisinga moiety to be linked to the bead.

In some embodiments, a degradable bead can be introduced into apartition, such that the bead degrades within the partition and anyassociated species (e.g., oligonucleotides) are released within thedroplet when the appropriate stimulus is applied. The free species(e.g., oligonucleotides, nucleic acid molecules) can interact with otherreagents contained in the partition. For example, a polyacrylamide beadfeaturing cystamine and linked, via a disulfide bond, to a barcodesequence, can be combined with a reducing agent within a droplet of awater-in-oil emulsion. Within the droplet, the reducing agent can breakthe various disulfide bonds, resulting in bead degradation and releaseof the barcode sequence into the aqueous, inner environment of thedroplet. In another example, heating of a droplet with a bead-boundbarcode sequence in basic solution can also result in bead degradationand release of the attached barcode sequence into the aqueous, innerenvironment of the droplet.

Any suitable number of species (e.g., primer, barcoded oligonucleotide)can be associated with a bead such that, upon release from the bead, thespecies (e.g., primer, e.g., barcoded oligonucleotide) are present inthe partition at a pre-defined concentration. Such pre-definedconcentration can be selected to facilitate certain reactions forgenerating a sequencing library, e.g., amplification, within thepartition. In some cases, the pre-defined concentration of the primercan be limited by the process of producing nucleic acid molecule (e.g.,oligonucleotide) bearing beads.

A degradable bead can include one or more species with a labile bondsuch that, when the bead/species is exposed to the appropriate stimulus,the bond is broken and the bead degrades. The labile bond can be achemical bond (e.g., covalent bond, ionic bond) or can be another typeof physical interaction (e.g., van der Waals interactions, dipole-dipoleinteractions, etc.). In some embodiments, a crosslinker used to generatea bead can include a labile bond. Upon exposure to the appropriateconditions, the labile bond can be broken and the bead degraded. Forexample, upon exposure of a polyacrylamide gel bead that includescystamine crosslinkers to a reducing agent, the disulfide bonds of thecystamine can be broken and the bead degraded. A degradable bead can beuseful in more quickly releasing an attached species (e.g., a nucleicacid molecule, a barcode sequence, a primer, etc.) from the bead whenthe appropriate stimulus is applied to the bead as compared to a beadthat does not degrade. For example, for a species bound to an innersurface of a porous bead or in the case of an encapsulated species, thespecies can have greater mobility and accessibility to other species insolution upon degradation of the bead. In some embodiments, a speciescan also be attached to a degradable bead via a degradable linker (e.g.,disulfide linker). The degradable linker can respond to the same stimulias the degradable bead or the two degradable species can respond todifferent stimuli. For example, a barcode sequence can be attached, viaa disulfide bond, to a polyacrylamide bead comprising cystamine. Uponexposure of the barcoded-bead to a reducing agent, the bead degrades andthe barcode sequence is released upon breakage of both the disulfidelinkage between the barcode sequence and the bead and the disulfidelinkages of the cystamine in the bead.

As will be appreciated from the above description, while referred to asdegradation of a bead, in many embodiments, degradation can refer to thedisassociation of a bound or entrained species from a bead, both withand without structurally degrading the physical bead itself. Forexample, entrained species can be released from beads through osmoticpressure differences due to, for example, changing chemicalenvironments. By way of example, alteration of bead pore sizes due toosmotic pressure differences can generally occur without structuraldegradation of the bead itself. In some cases, an increase in pore sizedue to osmotic swelling of a bead can permit the release of entrainedspecies within the bead. In some embodiments, osmotic shrinking of abead can cause a bead to better retain an entrained species due to poresize contraction. Numerous chemical triggers can be used to trigger thedegradation of beads within partitions. Examples of these chemicalchanges can include, but are not limited to pH-mediated changes to theintegrity of a component within the bead, degradation of a component ofa bead via cleavage of cross-linked bonds, and depolymerization of acomponent of a bead.

In some embodiments, a bead can be formed from materials that includedegradable chemical cross-linkers, such as BAC or cystamine. Degradationof such degradable cross-linkers can be accomplished through a number ofmechanisms. In some examples, a bead can be contacted with a chemicaldegrading agent that can induce oxidation, reduction or other chemicalchanges. For example, a chemical degrading agent can be a reducingagent, such as dithiothreitol (DTT). Additional examples of reducingagents can include β-mercaptoethanol, (2S)-2-amino-1,4-dimercaptobutane(dithiobutylamine or DTBA), tris(2-carboxyethyl) phosphine (TCEP), orcombinations thereof. A reducing agent can degrade the disulfide bondsformed between gel precursors forming the bead, and thus, degrade thebead.

In certain embodiments, a change in pH of a solution, such as anincrease in pH, can trigger degradation of a bead. In other embodiments,exposure to an aqueous solution, such as water, can trigger hydrolyticdegradation, and thus degradation of the bead. In some cases, anycombination of stimuli can trigger degradation of a bead. For example, achange in pH can enable a chemical agent (e.g., DTT) to become aneffective reducing agent. Beads can also be induced to release theircontents upon the application of a thermal stimulus. A change intemperature can cause a variety of changes to a bead. For example, heatcan cause a solid bead to liquefy. A change in heat can cause melting ofa bead such that a portion of the bead degrades. In other cases, heatcan increase the internal pressure of the bead components such that thebead ruptures or explodes. Heat can also act upon heat-sensitivepolymers used as materials to construct beads.

In addition to beads and analytes, partitions that are formed caninclude a variety of different reagents and species. For example, whenlysis reagents are present within the partitions, the lysis reagents canfacilitate the release of analytes within the partition. Examples oflysis agents include bioactive reagents, such as lysis enzymes that areused for lysis of different cell types, e.g., gram positive or negativebacteria, plants, yeast, mammalian, etc., such as lysozymes,achromopeptidase, lysostaphin, labiase, kitalase, lyticase, and avariety of other lysis enzymes available from, e.g., Sigma-Aldrich, Inc.(St. Louis, MO), as well as other commercially available lysis enzymes.Other lysis agents can additionally or alternatively be co-partitionedto cause the release analytes into the partitions. For example, in somecases, surfactant-based lysis solutions can be used to lyse cells,although these can be less desirable for emulsion based systems wherethe surfactants can interfere with stable emulsions. In someembodiments, lysis solutions can include non-ionic surfactants such as,for example, TritonX-100 and Tween 20. In some embodiments, lysissolutions can include ionic surfactants such as, for example, sarcosyland sodium dodecyl sulfate (SDS). Electroporation, thermal, acoustic ormechanical cellular disruption can also be used in certain embodiments,e.g., non-emulsion based partitioning such as encapsulation of analytesthat can be in addition to or in place of droplet partitioning, whereany pore size of the encapsulate is sufficiently small to retain nucleicacid fragments of a given size, following cellular disruption.

Examples of other species that can be co-partitioned with analytes inthe partitions include, but are not limited to, DNase and RNaseinactivating agents or inhibitors, such as proteinase K, chelatingagents, such as EDTA, and other reagents employed in removing orotherwise reducing negative activity or impact of different cell lysatecomponents on subsequent processing of nucleic acids. Additionalreagents can also be co-partitioned, including endonucleases to fragmentDNA, DNA polymerase enzymes and dNTPs used to amplify nucleic acidfragments and to attach the barcode molecular tags to the amplifiedfragments. Additional reagents can also include reverse transcriptaseenzymes, including enzymes with terminal transferase activity, primersand oligonucleotides, and switch oligonucleotides (also referred toherein as “switch oligos” or “template switching oligonucleotides”)which can be used for template switching. In some embodiments, templateswitching can be used to increase the length of a cDNA. Templateswitching can be used to append a predefined nucleic acid sequence tothe cDNA. In an example of template switching, cDNA can be generatedfrom reverse transcription of a template, e.g., cellular mRNA, where areverse transcriptase with terminal transferase activity can addadditional nucleotides, e.g., poly(C), to the cDNA in a templateindependent manner. Switch oligos can include sequences complementary tothe additional nucleotides, e.g., poly(G). The additional nucleotides(e.g., poly(C)) on the cDNA can hybridize to the additional nucleotides(e.g., poly(G)) on the switch oligo, whereby the switch oligo can beused by the reverse transcriptase as template to further extend thecDNA. Template switching oligonucleotides can include a hybridizationregion and a template region. The hybridization region can include anysequence capable of hybridizing to the target. In some cases, thehybridization region includes a series of G bases to complement theoverhanging C bases at the 3′ end of a cDNA molecule. The series of Gbases can include 1 G base, 2 G bases, 3 G bases, 4 G bases, 5 G basesor more than 5 G bases. The template sequence can include any sequenceto be incorporated into the cDNA. In some cases, the template regionincludes at least 1 (e.g., at least 2, 3, 4, 5 or more) tag sequencesand/or functional sequences. Switch oligos can include deoxyribonucleicacids; ribonucleic acids; modified nucleic acids including2-Aminopurine, 2,6-Diaminopurine (2-Amino-dA), inverted dT, 5-Methyl dC,2′-deoxyInosine, Super T (5-hydroxybutynl-2′-deoxyuridine), Super G(8-aza-7-deazaguanosine), locked nucleic acids (LNAs), unlocked nucleicacids (UNAs, e.g., UNA-A, UNA-U, UNA-C, UNA-G), Iso-dG, Iso-dC, 2′Fluoro bases (e.g., Fluoro C, Fluoro U, Fluoro A, and Fluoro G), andcombinations of the foregoing.

In some embodiments, beads that are partitioned with the analyte caninclude different types of oligonucleotides bound to the bead, where thedifferent types of oligonucleotides bind to different types of analytes.For example, a bead can include one or more first oligonucleotides(which can be capture probes, for example) that can bind or hybridize toa first type of analyte, such as mRNA for example, and one or moresecond oligonucleotides (which can be capture probes, for example) thatcan bind or hybridize to a second type of analyte, such as gDNA forexample. Partitions can also include lysis agents that aid in releasingnucleic acids from the co-partitioned cell, and can also include anagent (e.g., a reducing agent) that can degrade the bead and/or breakcovalent linkages between the oligonucleotides and the bead, releasingthe oligonucleotides into the partition. The released barcodedoligonucleotides (which can also be barcoded) can hybridize with mRNAreleased from the cell and also with gDNA released from the cell.

Barcoded constructs thus formed from hybridization can include a firsttype of construct that includes a sequence corresponding to an originalbarcode sequence from the bead and a sequence corresponding to atranscript from the cell, and a second type of construct that includes asequence corresponding to the original barcode sequence from the beadand a sequence corresponding to genomic DNA from the cell. The barcodedconstructs can then be released/removed from the partition and, in someembodiments, further processed to add any additional sequences. Theresulting constructs can then be sequenced, the sequencing dataprocessed, and the results used to spatially characterize the mRNA andthe gDNA from the cell.

In another example, a partition includes a bead that includes a firsttype of oligonucleotide (e.g., a first capture probe) with a firstbarcode sequence, a poly(T) priming sequence that can hybridize with thepoly(A) tail of an mRNA transcript, and a UMI barcode sequence that canuniquely identify a given transcript. The bead also includes a secondtype of oligonucleotide (e.g., a second capture probe) with a secondbarcode sequence, a targeted priming sequence that is capable ofspecifically hybridizing with a third barcoded oligonucleotide (e.g., ananalyte capture agent) coupled to an antibody that is bound to thesurface of the partitioned cell. The third barcoded oligonucleotideincludes a UMI barcode sequence that uniquely identifies the antibody(and thus, the particular cell surface feature to which it is bound).

In this example, the first and second barcoded oligonucleotides includethe same spatial barcode sequence (e.g., the first and second barcodesequences are the same), which permits downstream association ofbarcoded nucleic acids with the partition. In some embodiments, however,the first and second barcode sequences are different.

The partition also includes lysis agents that aid in releasing nucleicacids from the cell and can also include an agent (e.g., a reducingagent) that can degrade the bead and/or break a covalent linkage betweenthe barcoded oligonucleotides and the bead, releasing them into thepartition. The first type of released barcoded oligonucleotide canhybridize with mRNA released from the cell and the second type ofreleased barcoded oligonucleotide can hybridize with the third type ofbarcoded oligonucleotide, forming barcoded constructs.

The first type of barcoded construct includes a spatial barcode sequencecorresponding to the first barcode sequence from the bead and a sequencecorresponding to the UMI barcode sequence from the first type ofoligonucleotide, which identifies cell transcripts. The second type ofbarcoded construct includes a spatial barcode sequence corresponding tothe second barcode sequence from the second type of oligonucleotide, anda UMI barcode sequence corresponding to the third type ofoligonucleotide (e.g., the analyte capture agent) and used to identifythe cell surface feature. The barcoded constructs can then bereleased/removed from the partition and, in some embodiments, furtherprocessed to add any additional sequences. The resulting constructs arethen sequenced, sequencing data processed, and the results used tocharacterize the mRNA and cell surface feature of the cell.

The foregoing discussion involves two specific examples of beads witholigonucleotides for analyzing two different analytes within apartition. More generally, beads that are partitioned can have any ofthe structures described previously, and can include any of thedescribed combinations of oligonucleotides for analysis of two or more(e.g., three or more, four or more, five or more, six or more, eight ormore, ten or more, 12 or more, 15 or more, 20 or more, 25 or more, 30 ormore, 40 or more, 50 or more) different types of analytes within apartition. Examples of beads with combinations of different types ofoligonucleotides (e.g., capture probes) for concurrently analyzingdifferent combinations of analytes within partitions include, but arenot limited to: (a) genomic DNA and cell surface features (e.g., usingthe analyte capture agents described herein); (b) mRNA and a lineagetracing construct; (c) mRNA and cell methylation status; (d) mRNA andaccessible chromatin (e.g., ATAC-seq, DNase-seq, and/or MNase-seq); (e)mRNA and cell surface or intracellular proteins and/or metabolites; (f)a barcoded analyte capture agent (e.g., the MHC multimers describedherein) and a V(D)J sequence of an immune cell receptor (e.g., T-cellreceptor); and (g) mRNA and a perturbation agent (e.g., a CRISPRcrRNA/sgRNA, TALEN, zinc finger nuclease, and/or antisenseoligonucleotide as described herein).

(F) Sequencing Analysis

After analytes from the sample have hybridized or otherwise beenassociated with capture probes, analyte capture agents, or otherbarcoded oligonucleotide sequences according to any of the methodsdescribed above in connection with the general spatial cell-basedanalytical methodology, the barcoded constructs that result fromhybridization/association are analyzed via sequencing to identify theanalytes.

In some embodiments, where a sample is barcoded directly viahybridization with capture probes or analyte capture agents hybridized,bound, or associated with either the cell surface, or introduced intothe cell, as described above, sequencing can be performed on the intactsample. Alternatively, if the barcoded sample has been separated intofragments, cell groups, or individual cells, as described above,sequencing can be performed on individual fragments, cell groups, orcells. For analytes that have been barcoded via partitioning with beads,as described above, individual analytes (e.g., cells, or cellularcontents following lysis of cells) can be extracted from the partitionsby breaking the partitions, and then analyzed by sequencing to identifythe analytes.

A wide variety of different sequencing methods can be used to analyzebarcoded analyte constructs. In general, sequenced polynucleotides canbe, for example, nucleic acid molecules such as deoxyribonucleic acid(DNA) or ribonucleic acid (RNA), including variants or derivativesthereof (e.g., single stranded DNA or DNA/RNA hybrids, and nucleic acidmolecules with a nucleotide analog).

Sequencing of polynucleotides can be performed by various commercialsystems. More generally, sequencing can be performed using nucleic acidamplification, polymerase chain reaction (PCR) (e.g., digital PCR anddroplet digital PCR (ddPCR), quantitative PCR, real time PCR, multiplexPCR, PCR-based singleplex methods, emulsion PCR), and/or isothermalamplification.

Other examples of methods for sequencing genetic material include, butare not limited to, DNA hybridization methods (e.g., Southern blotting),restriction enzyme digestion methods, Sanger sequencing methods,next-generation sequencing methods (e.g., single-molecule real-timesequencing, nanopore sequencing, and Polony sequencing), ligationmethods, and microarray methods. Additional examples of sequencingmethods that can be used include targeted sequencing, single moleculereal-time sequencing, exon sequencing, electron microscopy-basedsequencing, panel sequencing, transistor-mediated sequencing, directsequencing, random shotgun sequencing, Sanger dideoxy terminationsequencing, whole-genome sequencing, sequencing by hybridization,pyrosequencing, capillary electrophoresis, gel electrophoresis, duplexsequencing, cycle sequencing, single-base extension sequencing,solid-phase sequencing, high-throughput sequencing, massively parallelsignature sequencing, co-amplification at lower denaturationtemperature-PCR (COLD-PCR), sequencing by reversible dye terminator,paired-end sequencing, near-term sequencing, exonuclease sequencing,sequencing by ligation, short-read sequencing, single-moleculesequencing, sequencing-by-synthesis, real-time sequencing,reverse-terminator sequencing, nanopore sequencing, 454 sequencing,Solexa Genome Analyzer sequencing, SOLiD™ sequencing, MS-PET sequencing,and any combinations thereof.

Sequence analysis of the nucleic acid molecules (including barcodednucleic acid molecules or derivatives thereof) can be direct orindirect. Thus, the sequence analysis substrate (which can be viewed asthe molecule which is subjected to the sequence analysis step orprocess) can directly be the barcoded nucleic acid molecule or it can bea molecule which is derived therefrom (e.g., a complement thereof).Thus, for example, in the sequence analysis step of a sequencingreaction, the sequencing template can be the barcoded nucleic acidmolecule or it can be a molecule derived therefrom. For example, a firstand/or second strand DNA molecule can be directly subjected to sequenceanalysis (e.g. sequencing), i.e., can directly take part in the sequenceanalysis reaction or process (e.g. the sequencing reaction or sequencingprocess, or be the molecule which is sequenced or otherwise identified).Alternatively, the barcoded nucleic acid molecule can be subjected to astep of second strand synthesis or amplification before sequenceanalysis (e.g. sequencing or identification by another technique). Thesequence analysis substrate (e.g., template) can thus be an amplicon ora second strand of a barcoded nucleic acid molecule.

In some embodiments, both strands of a double stranded molecule can besubjected to sequence analysis (e.g., sequenced). In some embodiments,single stranded molecules (e.g. barcoded nucleic acid molecules) can beanalyzed (e.g. sequenced). To perform single molecule sequencing, thenucleic acid strand can be modified at the 3′ end.

Massively parallel sequencing techniques can be used for sequencingnucleic acids, as described above. In one embodiment, a massivelyparallel sequencing technique can be based on reversibledye-terminators. As an example, DNA molecules are first attached toprimers on, e.g., a glass or silicon substrate, and amplified so thatlocal clonal colonies are formed (bridge amplification). Four types ofddNTPs are added, and non-incorporated nucleotides are washed away.Unlike pyrosequencing, the DNA is only extended one nucleotide at a timedue to a blocking group (e.g., 3′ blocking group present on the sugarmoiety of the ddNTP). A detector acquires images of the fluorescentlylabelled nucleotides, and then the dye along with the terminal 3′blocking group is chemically removed from the DNA, as a precursor to asubsequent cycle. This process can be repeated until the requiredsequence data is obtained.

As another example, massively parallel pyrosequencing techniques canalso be used for sequencing nucleic acids. In pyrosequencing, thenucleic acid is amplified inside water droplets in an oil solution(emulsion PCR), with each droplet containing a single nucleic acidtemplate attached to a single primer-coated bead that then forms aclonal colony. The sequencing system contains many picolitre-volumewells each containing a single bead and sequencing enzymes.Pyrosequencing uses luciferase to generate light for detection of theindividual nucleotides added to the nascent nucleic acid and thecombined data are used to generate sequence reads.

As another example application of pyrosequencing, released PPi can bedetected by being immediately converted to adenosine triphosphate (ATP)by ATP sulfurylase, and the level of ATP generated can be detected vialuciferase-produced photons, such as described in Ronaghi, et al., Anal.Biochem. 242(1), 84-9 (1996); Ronaghi, Genome Res. 11(1), 3-11 (2001);Ronaghi et al. Science 281 (5375), 363 (1998); and U.S. Pat. Nos.6,210,891, 6,258,568, and 6,274,320, the entire contents of each ofwhich are incorporated herein by reference.

In some embodiments, sequencing is performed by detection of hydrogenions that are released during the polymerization of DNA. A microwellcontaining a template DNA strand to be sequenced can be flooded with asingle type of nucleotide. If the introduced nucleotide is complementaryto the leading template nucleotide, it is incorporated into the growingcomplementary strand. This causes the release of a hydrogen ion thattriggers a hypersensitive ion sensor, which indicates that a reactionhas occurred. If homopolymer repeats are present in the templatesequence, multiple nucleotides will be incorporated in a single cycle.This leads to a corresponding number of released hydrogen ions and aproportionally higher electronic signal.

In some embodiments, sequencing can be performed in-situ. In-situsequencing methods are particularly useful, for example, when thebiological sample remains intact after analytes on the sample surface(e.g., cell surface analytes) or within the sample (e.g., intracellularanalytes) have been barcoded. In-situ sequencing typically involvesincorporation of a labeled nucleotide (e.g., fluorescently labeledmononucleotides or dinucleotides) in a sequential, template-dependentmanner or hybridization of a labeled primer (e.g., a labeled randomhexamer) to a nucleic acid template such that the identities (i.e.,nucleotide sequence) of the incorporated nucleotides or labeled primerextension products can be determined, and consequently, the nucleotidesequence of the corresponding template nucleic acid. Aspects of in-situsequencing are described, for example, in Mitra et al., (2003) Anal.Biochem., 320, 55-65, and Lee et al., (2014) Science, 343(6177),1360-1363, the entire contents of each of which are incorporated hereinby reference.

In addition, examples of methods and systems for performing in-situsequencing are described in PCT Patent Application Publication Nos.WO2014/163886, WO2018/045181, W02018/045186, and in U.S. Pat. Nos.10,138,509 and 10,179,932, the entire contents of each of which areincorporated herein by reference. Example techniques for in-situsequencing include, but are not limited to, STARmap (described forexample in Wang et al., (2018) Science, 361(6499) 5691), MERFISH(described for example in Moffitt, (2016) Methods in Enzymology, 572,1-49), and FISSEQ (described for example in U.S. Pat. ApplicationPublication No. 2019/0032121). The entire contents of each of theforegoing references are incorporated herein by reference.

For analytes that have been barcoded via partitioning, barcoded nucleicacid molecules or derivatives thereof (e.g., barcoded nucleic acidmolecules to which one or more functional sequences have been added, orfrom which one or more features have been removed) can be pooled andprocessed together for subsequent analysis such as sequencing on highthroughput sequencers. Processing with pooling can be implemented usingbarcode sequences. For example, barcoded nucleic acid molecules of agiven partition can have the same barcode, which is different frombarcodes of other spatial partitions. Alternatively, barcoded nucleicacid molecules of different partitions can be processed separately forsubsequent analysis (e.g., sequencing).

In some embodiments, where capture probes do not contain a spatialbarcode, the spatial barcode can be added after the capture probecaptures analytes from a biological sample and before analysis of theanalytes. When a spatial barcode is added after an analyte is captured,the barcode can be added after amplification of the analyte (e.g.,reverse transcription and polymerase amplification of RNA). In someembodiments, analyte analysis uses direct sequencing of one or morecaptured analytes, such as direct sequencing of hybridized RNA. In someembodiments, direct sequencing is performed after reverse transcriptionof hybridized RNA. In some embodiments direct sequencing is performedafter amplification of reverse transcription of hybridized RNA.

In some embodiments, direct sequencing of captured RNA is performed bysequencing-by-synthesis (SBS). In some embodiments, a sequencing primeris complementary to a sequence in one or more of the domains of acapture probe (e.g., functional domain). In such embodiments,sequencing-by-synthesis can include reverse transcription and/oramplification in order to generate a template sequence (e.g., functionaldomain) from which a primer sequence can bind.

SBS can involve hybridizing an appropriate primer, sometimes referred toas a sequencing primer, with the nucleic acid template to be sequenced,extending the primer, and detecting the nucleotides used to extend theprimer. Preferably, the nucleic acid used to extend the primer isdetected before a further nucleotide is added to the growing nucleicacid chain, thus allowing base-by-base in situ nucleic acid sequencing.The detection of incorporated nucleotides is facilitated by includingone or more labelled nucleotides in the primer extension reaction. Toallow the hybridization of an appropriate sequencing primer to thenucleic acid template to be sequenced, the nucleic acid template shouldnormally be in a single stranded form. If the nucleic acid templatesmaking up the nucleic acid spots are present in a double stranded formthese can be processed to provide single stranded nucleic acid templatesusing methods well known in the art, for example by denaturation,cleavage etc. The sequencing primers which are hybridized to the nucleicacid template and used for primer extension are preferably shortoligonucleotides, for example, 15 to 25 nucleotides in length. Thesequencing primers can be greater than 25 nucleotides in length as well.For example, sequencing primers can be about 20 to about 60 nucleotidesin length, or more than 60 nucleotides in length. The sequencing primerscan be provided in solution or in an immobilized form. Once thesequencing primer has been annealed to the nucleic acid template to besequenced by subjecting the nucleic acid template and sequencing primerto appropriate conditions, primer extension is carried out, for exampleusing a nucleic acid polymerase and a supply of nucleotides, at leastsome of which are provided in a labelled form, and conditions suitablefor primer extension if a suitable nucleotide is provided.

Preferably after each primer extension step, a washing step is includedin order to remove unincorporated nucleotides which can interfere withsubsequent steps. Once the primer extension step has been carried out,the nucleic acid colony is monitored to determine whether a labellednucleotide has been incorporated into an extended primer. The primerextension step can then be repeated to determine the next and subsequentnucleotides incorporated into an extended primer. If the sequence beingdetermined is unknown, the nucleotides applied to a given colony areusually applied in a chosen order which is then repeated throughout theanalysis, for example dATP, dTTP, dCTP, dGTP.

SBS techniques which can be used are described for example, but notlimited to, those in U.S. Pat. App. Pub. No. 2007/0166705, U.S. Pat.App. Pub. No. 2006/0188901, U.S. Pat. 7,057,026, U.S. Pat. App. Pub. No.2006/0240439, U.S. Pat. App. Pub. No. 2006/0281109, PCT Patent App. Pub.No. WO 05/065814, U.S. Pat. App. Pub. No. 2005/0100900, PCT Patent App.Pub. No. WO 06/064199, PCT Patent App. Pub. No. WO07/010,251, U.S. Pat.App. Pub. No. 2012/0270305, U.S. Pat. App. Pub. No. 2013/0260372, andU.S. Pat. App. Pub. No. 2013/0079232, the entire contents of each ofwhich are incorporated herein by reference.

In some embodiments, direct sequencing of captured RNA is performed bysequential fluorescence hybridization (e.g., sequencing byhybridization). In some embodiments, a hybridization reaction where RNAis hybridized to a capture probe is performed in situ. In someembodiments, captured RNA is not amplified prior to hybridization with asequencing probe. In some embodiments, RNA is amplified prior tohybridization with sequencing probes (e.g., reverse transcription tocDNA and amplification of cDNA). In some embodiments, amplification isperformed using single-molecule hybridization chain reaction. In someembodiments, amplification is performed using rolling chainamplification.

Sequential fluorescence hybridization can involve sequentialhybridization of probes including degenerate primer sequences and adetectable label. A degenerate primer sequence is a shortoligonucleotide sequence which is capable of hybridizing to any nucleicacid fragment independent of the sequence of said nucleic acid fragment.For example, such a method could include the steps of: (a) providing amixture including four probes, each of which includes either A, C, G, orT at the 5′-terminus, further including degenerate nucleotide sequenceof 5 to 11 nucleotides in length, and further including a functionaldomain (e.g., fluorescent molecule) that is distinct for probes with A,C, G, or T at the 5′-terminus; (b) associating the probes of step (a) tothe target polynucleotide sequences, whose sequence needs will bedetermined by this method; (c) measuring the activities of the fourfunctional domains and recording the relative spatial location of theactivities; (d) removing the reagents from steps (a)-(b) from the targetpolynucleotide sequences; and repeating steps (a)-(d) for n cycles,until the nucleotide sequence of the spatial domain for each bead isdetermined, with modification that the oligonucleotides used in step (a)are complementary to part of the target polynucleotide sequences and thepositions 1 through n flanking the part of the sequences. Because thebarcode sequences are different, in some embodiments, these additionalflanking sequences are degenerate sequences. The fluorescent signal fromeach spot on the array for cycles 1 through n can be used to determinethe sequence of the target polynucleotide sequences.

In some embodiments, direct sequencing of captured RNA using sequentialfluorescence hybridization is performed in vitro. In some embodiments,captured RNA is amplified prior to hybridization with a sequencing probe(e.g., reverse transcription to cDNA and amplification of cDNA). In someembodiments, a capture probe containing captured RNA is exposed to thesequencing probe targeting coding regions of RNA. In some embodiments,one or more sequencing probes are targeted to each coding region. Insome embodiments, the sequencing probe is designed to hybridize withsequencing reagents (e.g., a dye-labeled readout oligonucleotides). Asequencing probe can then hybridize with sequencing reagents. In someembodiments, output from the sequencing reaction is imaged. In someembodiments, a specific sequence of cDNA is resolved from an image of asequencing reaction. In some embodiments, reverse transcription ofcaptured RNA is performed prior to hybridization to the sequencingprobe. In some embodiments, the sequencing probe is designed to targetcomplementary sequences of the coding regions of RNA (e.g., targetingcDNA).

In some embodiments, a captured RNA is directly sequenced using ananopore-based method. In some embodiments, direct sequencing isperformed using nanopore direct RNA sequencing in which captured RNA istranslocated through a nanopore. A nanopore current can be recorded andconverted into a base sequence. In some embodiments, captured RNAremains attached to a substrate during nanopore sequencing. In someembodiments, captured RNA is released from the substrate prior tonanopore sequencing. In some embodiments, where the analyte of interestis a protein, direct sequencing of the protein can be performed usingnanopore-based methods. Examples of nanopore-based sequencing methodsthat can be used are described in Deamer et al., Trends Biotechnol. 18,14 7-151 (2000); Deamer et al., Acc. Chem. Res. 35:817-825 (2002); Li etal., Nat. Mater. 2:611-615 (2003); Soni et al., Clin. Chem. 53,1996-2001 (2007); Healy et al., Nanomed. 2, 459-481 (2007); Cockroft etal., J. Am. Chem. Soc. 130, 818-820 (2008); and in U.S. Pat. 7,001,792.The entire contents of each of the foregoing references are incorporatedherein by reference.

In some embodiments, direct sequencing of captured RNA is performedusing single molecule sequencing by ligation. Such techniques utilizeDNA ligase to incorporate oligonucleotides and identify theincorporation of such oligonucleotides. The oligonucleotides typicallyhave different labels that are correlated with the identity of aparticular nucleotide in a sequence to which the oligonucleotideshybridize. Aspects and features involved in sequencing by ligation aredescribed, for example, in Shendure et al. Science (2005), 309:1728-1732, and in U.S. Pat. Nos. 5,599,675; 5,750,341; 6,969,488;6,172,218; and 6,306,597, the entire contents of each of which areincorporated herein by reference.

In some embodiments, nucleic acid hybridization can be used forsequencing. These methods utilize labeled nucleic acid decoder probesthat are complementary to at least a portion of a barcode sequence.Multiplex decoding can be performed with pools of many different probeswith distinguishable labels. Non-limiting examples of nucleic acidhybridization sequencing are described for example in U.S. Pat. No.8,460,865, and in Gunderson et al., Genome Research 14:870-877 (2004),the entire contents of each of which are incorporated herein byreference.

In some embodiments, commercial high-throughput digital sequencingtechniques can be used to analyze barcode sequences, in which DNAtemplates are prepared for sequencing not one at a time, but in a bulkprocess, and where many sequences are read out preferably in parallel,or alternatively using an ultra-high throughput serial process thatitself may be parallelized. Examples of such techniques includeIllumina® sequencing (e.g., flow cell-based sequencing techniques),sequencing by synthesis using modified nucleotides (such ascommercialized in TruSeq™ and HiSeq™ technology by Illumina, Inc., SanDiego, CA), HeliScope™ by Helicos Biosciences Corporation, Cambridge,MA, and PacBio RS by Pacific Biosciences of California, Inc., MenloPark, CA), sequencing by ion detection technologies (Ion Torrent, Inc.,South San Francisco, CA), and sequencing of DNA nanoballs (CompleteGenomics, Inc., Mountain View, CA).

In some embodiments, detection of a proton released upon incorporationof a nucleotide into an extension product can be used in the methodsdescribed herein. For example, the sequencing methods and systemsdescribed in U.S. Pat. Application Publication Nos. 2009/0026082,2009/0127589, 2010/0137143, and 2010/0282617, can be used to directlysequence barcodes. The entire contents of each of the foregoingreferences are incorporated herein by reference.

In some embodiments, real-time monitoring of DNA polymerase activity canbe used during sequencing. For example, nucleotide incorporations can bedetected through fluorescence resonance energy transfer (FRET), asdescribed for example in Levene et al., Science (2003), 299, 682-686,Lundquist et al., Opt. Lett. (2008), 33, 1026-1028, and Korlach et al.,Proc. Natl. Acad. Sci. USA (2008), 105, 1176-1181. The entire contentsof each of the foregoing references are herein incorporated byreference.

IV. Multiplexing (A) Multiplexing Generally

In various embodiments of spatial analysis as described herein, featurescan include different types of capture probes for analyzing bothintrinsic and extrinsic information for individual cells. For example, afeature can include one or more of the following: 1) a capture probefeaturing a capture domain that binds to one or more endogenous nucleicacids in the cell; 2) a capture probe featuring a capture domain thatbinds to one or more exogenous nucleic acids in the cell (e.g., nucleicacids from a microorganism (e.g., a virus, a bacterium)) that infectsthe cell, nucleic acids introduced into the cell (e.g., such as plasmidsor nucleic acid derived therefrom), nucleic acids for gene editing(e.g., CRISPR-related RNA such as crRNA, guide RNA); 3) a capture probefeaturing a capture domain that binds to a analyte capture agent (e.g.,an antibody coupled to a oligonucleotide that includes a capture agentbarcode domain having an analyte capture sequence that binds the capturedomain), and 4) a capture moiety featuring a domain that binds to aprotein (e.g., an exogenous protein expressed in the cell, a proteinfrom a microorganism (e.g., a virus, a bacterium)) that infects thecell, or a binding partner for a protein of the cell (e.g., an antigenfor an immune cell receptor).

In some embodiments of any of the spatial analysis methods as describedherein, spatial profiling includes concurrent analysis of two differenttypes of analytes. A feature can be a gel bead, which is coupled (e.g.,reversibly coupled) to one or more capture probes. The capture probescan include a spatial barcode sequence and a poly (T) priming sequencethat can hybridize with the poly (A) tail of an mRNA transcript. Thecapture probe can also include a UMI sequence that can uniquely identifya given transcript. The capture probe can also include a spatial barcodesequence and a random N-mer priming sequence that is capable of randomlyhybridizing with gDNA. In this configuration, capture probes can includethe same spatial barcode sequence, which permits association ofdownstream sequencing reads with the feature.

In some embodiments of any of the spatial analysis methods as describedherein, a feature can be a gel bead, which is coupled (e.g., reversiblycoupled) to capture probes. The Capture probe can include a spatialbarcode sequence and a poly(T) priming sequence 614 that can hybridizewith the poly(A) tail of an mRNA transcript. The capture probe can alsoinclude a UMI sequence that can uniquely identify a given transcript.The capture probe can include a spatial barcode sequence and a capturedomain that is capable of specifically hybridizing with an analytecapture agent. The analyte capture agent can includes an oligonucleotidethat includes an analyte capture sequence that interacts with thecapture domain coupled to the feature. The oligonucleotide of theanalyte capture agent can be coupled to an antibody that is bound to thesurface of a cell. The oligonucleotide includes a barcode sequence(e.g., an analyte binding moiety barcode) that uniquely identifies theantibody (and thus, the particular cell surface feature to which it isbound). In this configuration, the capture probes include the samespatial barcode sequence, which permit downstream association ofbarcoded nucleic acids with the location on the spatial array. In someembodiments of any of the spatial profiling methods described herein,the analyte capture agents can be can be produced by any suitable route,including via example coupling schemes described elsewhere herein.

In some embodiments of any of the spatial analysis methods describedherein, other combinations of two or more biological analytes that canbe concurrently measured include, without limitation: (a) genomic DNAand cell surface features (e.g., via analyte capture agents that bind toa cell surface feature), (b) mRNA and a lineage tracing construct, (c)mRNA and cell methylation status, (d) mRNA and accessible chromatin(e.g., ATAC-seq, DNase-seq, and/or MNase-seq), (e) mRNA and cell surfaceor intracellular proteins and/or metabolites, (f) mRNA and chromatin(spatial organization of chromatin in a cell), (g) an analyte captureagent (e.g., any of the MHC multimers described herein) and a V(D)Jsequence of an immune cell receptor (e.g., T-cell receptor), (h) mRNAand a perturbation agent (e.g., a CRISPR crRNA/sgRNA, TALEN, zinc fingernuclease, and/or antisense oligonucleotide as described herein), (i)genomic DNA and a perturbation agent, (j) an analyte capture agent and aperturbation reagents, (k) accessible chromatin and a perturbationreagent, (1) chromatin (e.g., spatial organization of chromatin in acell) and a perturbation reagent, and (m) cell surface or intracellularproteins and/or metabolites and a perturbation reagent, or anycombination thereof.

In some embodiments of any of the spatial analysis methods describedherein, the first analyte can include a nucleic acid molecule with anucleic acid sequence (e.g., mRNA, complementary DNA derived fromreverse transcription of mRNA) encoding at least a portion of a V(D)Jsequence of an immune cell receptor (e.g., a TCR or BCR). In someembodiments, the nucleic acid molecule with a nucleic acid sequenceencoding at least a portion of a V(D)J sequence of an immune cellreceptor is cDNA first generated from reverse transcription of thecorresponding mRNA, using a poly(T) containing primer. The cDNA that isgenerated can then be barcoded using a primer, featuring a spatialbarcode sequence (and optionally, a UMI sequence) that hybridizes withat least a portion of the cDNA that is generated. In some embodiments, atemplate switching oligonucleotide in conjunction a terminal transferaseor a reverse transcriptase having terminal transferase activity can beemployed to generate a priming region on the cDNA to which a barcodedprimer can hybridize during cDNA generation. Terminal transferaseactivity can, for example, add a poly(C) tail to a 3′ end of the cDNAsuch that the template switching oligonucleotide can bind via a poly(G)priming sequence and the 3′ end of the cDNA can be further extended. Theoriginal mRNA template and template switching oligonucleotide can thenbe denatured from the cDNA and the barcoded primer comprising a sequencecomplementary to at least a portion of the generated priming region onthe cDNA can then hybridize with the cDNA and a barcoded constructcomprising the barcode sequence (and any optional UMI sequence) and acomplement of the cDNA generated. Additional methods and compositionssuitable for barcoding cDNA generated from mRNA transcripts includingthose encoding V(D)J regions of an immune cell receptor and/or barcodingmethods and composition including a template switch oligonucleotide aredescribed, for example, in PCT Patent Application Publication No. WO2018/075693, and in U.S. Pat. Application Publication No. 2018/0105808,the entire contents of each of which are incorporated herein byreference.

In some embodiments, V(D)J analysis can be performed using methodssimilar to those described herein. For example, V(D)J analysis can becompleted with the use of one or more analyte capture agents that bindto particular surface features of immune cells and are associated withbarcode sequences (e.g., analyte binding moiety barcodes). The one ormore analyte capture agents can include an MHC or MHC multimer. Abarcoded oligonucleotide coupled to a bead that can be used for V(D)Janalysis. The oligonucleotide is coupled to a bead by a releasablelinkage, such as a disulfide linker. The oligonucleotide can includefunctional sequences that are useful for subsequent processing, such asfunctional sequence, which can include a sequencer specific flow cellattachment sequence, e.g., a P5 sequence, as well as functionalsequence, which can include sequencing primer sequences, e.g., a R1primer binding site. In some embodiments, the sequence can include a P7sequence and a R2 primer binding site. A barcode sequence can beincluded within the structure for use in barcoding the templatepolynucleotide. The functional sequences can be selected forcompatibility with a variety of different sequencing systems, e.g., 454Sequencing, Ion Torrent Proton or PGM, Illumina X10, etc., and therequirements thereof. In some embodiments, the barcode sequence,functional sequences (e.g., flow cell attachment sequence) andadditional sequences (e.g., sequencing primer sequences) can be commonto all of the oligonucleotides attached to a given bead. The barcodedoligonucleotide can also include a sequence to facilitate templateswitching (e.g., a poly(G) sequence). In some embodiments, theadditional sequence provides a unique molecular identifier (UMI)sequence segment, as described elsewhere herein.

In an exemplary method of cellular polynucleotide analysis using abarcode oligonucleotide, a cell is co-partitioned along with a beadbearing a barcoded oligonucleotide and additional reagents such as areverse transcriptase, primers, oligonucleotides (e.g., templateswitching oligonucleotides), dNTPs, and a reducing agent into apartition (e.g., a droplet in an emulsion). Within the partition, thecell can be lysed to yield a plurality of template polynucleotides(e.g., DNA such as genomic DNA, RNA such as mRNA, etc.).

A reaction mixture featuring a template polynucleotide from a cell and(i) the primer having a sequence towards a 3′ end that hybridizes to thetemplate polynucleotide (e.g., poly(T)) and (ii) a template switchingoligonucleotide that includes a first oligonucleotide towards a 5′ endcan be subjected to an amplification reaction to yield a firstamplification product. In some embodiments, the template polynucleotideis an mRNA with a poly(A) tail and the primer that hybridizes to thetemplate polynucleotide includes a poly(T) sequence towards a 3′ end,which is complementary to the poly(A) segment. The first oligonucleotidecan include at least one of an adaptor sequence, a barcode sequence, aunique molecular identifier (UMI) sequence, a primer binding site, and asequencing primer binding site or any combination thereof. In somecases, a first oligonucleotide is a sequence that can be common to allpartitions of a plurality of partitions. For example, the firstoligonucleotide can include a flow cell attachment sequence, anamplification primer binding site, or a sequencing primer binding siteand the first amplification reaction facilitates the attachment theoligonucleotide to the template polynucleotide from the cell. In someembodiments, the first oligonucleotide includes a primer binding site.In some embodiments, the first oligonucleotide includes a sequencingprimer binding site.

The sequence towards a 3′ end (e.g., poly(T)) of the primer hybridizesto the template polynucleotide. In a first amplification reaction,extension reaction reagents, e.g., reverse transcriptase, nucleosidetriphosphates, co-factors (e.g., Mg²⁺ or Mn²⁺), that are alsoco-partitioned, can extend the primer sequence using the cell’s nucleicacid as a template, to produce a transcript, e.g., cDNA, having afragment complementary to the strand of the cell’s nucleic acid to whichthe primer annealed. In some embodiments, the reverse transcriptase hasterminal transferase activity and the reverse transcriptase addsadditional nucleotides, e.g., poly(C), to the cDNA in a templateindependent manner.

The template switching oligonucleotide, for example a template switchingoligonucleotide which includes a poly(G) sequence, can hybridize to thecDNA and facilitate template switching in the first amplificationreaction. The transcript, therefore, can include the sequence of theprimer, a sequence complementary to the template polynucleotide from thecell, and a sequence complementary to the template switchingoligonucleotide.

In some embodiments of any of the spatial analysis methods describedherein, subsequent to the first amplification reaction, the firstamplification product or transcript can be subjected to a secondamplification reaction to generate a second amplification product. Insome embodiments, additional sequences (e.g., functional sequences suchas flow cell attachment sequence, sequencing primer binding sequences,barcode sequences, etc.) are attached. The first and secondamplification reactions can be performed in the same volume, such as forexample in a droplet. In some embodiments, the first amplificationproduct is subjected to a second amplification reaction in the presenceof a barcoded oligonucleotide to generate a second amplification producthaving a barcode sequence. The barcode sequence can be unique to apartition, that is, each partition can have a unique barcode sequence.The barcoded oligonucleotide can include a sequence of at least asegment of the template switching oligonucleotide and at least a secondoligonucleotide. The segment of the template switching oligonucleotideon the barcoded oligonucleotide can facilitate hybridization of thebarcoded oligonucleotide to the transcript, e.g., cDNA, to facilitatethe generation of a second amplification product. In addition to abarcode sequence, the barcoded oligonucleotide can include a secondoligonucleotide such as at least one of an adaptor sequence, a uniquemolecular identifier (UMI) sequence, a primer binding site, and asequencing primer binding site, or any combination thereof.

In some embodiments of any of the spatial analysis methods describedherein, the second amplification reaction uses the first amplificationproduct as a template and the barcoded oligonucleotide as a primer. Insome embodiments, the segment of the template switching oligonucleotideon the barcoded oligonucleotide can hybridize to the portion of the cDNAor complementary fragment having a sequence complementary to thetemplate switching oligonucleotide or that which was copied from thetemplate switching oligonucleotide. In the second amplificationreaction, extension reaction reagents, e.g., polymerase, nucleosidetriphosphates, co-factors (e.g., Mg²⁺ or Mn²⁺), that are alsoco-partitioned, can extend the primer sequence using the firstamplification product as template. The second amplification product caninclude a second oligonucleotide, a sequence of a segment of thetemplate polynucleotide (e.g., mRNA), and a sequence complementary tothe primer.

In some embodiments of any of the spatial analysis methods describedherein, the second amplification product uses the barcodedoligonucleotide as a template and at least a portion of the firstamplification product as a primer. The segment of the firstamplification product (e.g., cDNA) having a sequence complementary tothe template switching oligonucleotide can hybridize to the segment ofthe barcoded oligonucleotide comprising a sequence of at least a segmentof the template switching oligonucleotide. In the second amplificationreaction, extension reaction reagents, e.g., polymerase, nucleosidetriphosphates, co-factors (e.g., Mg²⁺or Mn²⁺), that are alsoco-partitioned, can extend the primer sequence (e.g., firstamplification product) using the barcoded oligonucleotide as template.The second amplification product can include the sequence of the primer,a sequence which is complementary to the sequence of the templatepolynucleotide (e.g., mRNA), and a sequence complementary to the secondoligonucleotide.

In some embodiments of any of the spatial analysis methods describedherein, three or more classes of biological analytes can be concurrentlymeasured. For example, a feature can include capture probes that canparticipate in an assay of at least three different types of analytesvia three different capture domains. A bead can be coupled to a barcodedoligonucleotide that includes a capture domain that includes a poly(T)priming sequence for mRNA analysis; a barcoded oligonucleotide thatincludes a capture domain that includes a random N-mer priming sequencefor gDNA analysis; and a barcoded oligonucleotide that includes acapture domain that can specifically bind a an analyte capture agent(e.g., an antibody with a spatial barcode), via its analyte capturesequence.

In some embodiments of any of the spatial analysis methods describedherein, other combinations of three or more biological analytes that canbe concurrently measured include, without limitation: (a) mRNA, alineage tracing construct, and cell surface and/or intracellularproteins and/or metabolites; (b) mRNA, accessible chromatin (e.g.,ATAC-seq, DNase-seq, and/or MNase-seq), and cell surface and/orintracellular proteins and/or metabolites; (c) mRNA, genomic DNA, and aperturbation reagent (e.g., a CRISPR crRNA/sgRNA, TALEN, zinc fingernuclease, and/or antisense oligonucleotide as described herein); (d)mRNA, accessible chromatin, and a perturbation reagent; (e) mRNA, ananalyte capture agent (e.g., any of the MHC multimers described herein),and a perturbation reagent; (f) mRNA, cell surface and/or intracellularproteins and/or metabolites, and a perturbation agent; (g) mRNA, a V(D)Jsequence of an immune cell receptor (e.g., T-cell receptor), and aperturbation reagent; (h) mRNA, an analyte capture agent, and a V(D)Jsequence of an immune cell receptor; (i) cell surface and/orintracellular proteins and/or metabolites, a an analyte capture agent(e.g., the MHC multimers described herein), and a V(D)J sequence of animmune cell receptor; (j) methylation status, mRNA, and cell surfaceand/or intracellular proteins and/or metabolites; (k) mRNA, chromatin(e.g., spatial organization of chromatin in a cell), and a perturbationreagent; (1) a V(D)J sequence of an immune cell receptor, chromatin(e.g., spatial organization of chromatin in a cell); and a perturbationreagent; and (m) mRNA, a V(D)J sequence of an immune cell receptor, andchromatin (e.g., spatial organization of chromatin in a cell), or anycombination thereof.

In some embodiments of any of the spatial analysis methods describedherein, four or more classes biological analytes can be concurrentlymeasured. A feature can be a bead that is coupled to barcoded primersthat can each participate in an assay of a different type of analyte.The feature is coupled (e.g., reversibly coupled) to a capture probethat includes a capture domain that includes a poly(T) priming sequencefor mRNA analysis and is also coupled (e.g., reversibly coupled) tocapture probe that includes a capture domain that includes a randomN-mer priming sequence for gDNA analysis. Moreover, the feature is alsocoupled (e.g., reversibly coupled) to a capture probe that binds ananalyte capture sequence of an analyte capture agent via its capturedomain. The feature can also be coupled (e.g., reversibly coupled) to acapture probe that can specifically bind a nucleic acid molecule thatcan function as a perturbation agent (e.g., a CRISPR crRNA/sgRNA, TALEN,zinc finger nuclease, and/or antisense oligonucleotide as describedherein), via its capture domain.

In some embodiments of any of the spatial analysis methods describedherein, each of the various spatially barcoded capture probes present ata given feature or on a given bead include the same spatial barcodesequence. In some embodiments, each barcoded capture probe can bereleased from the feature in a manner suitable for analysis of itsrespective analyte. For example, barcoded constructs A, B, C and D canbe generated as described elsewhere herein and analyzed. Barcodedconstruct A can include a sequence corresponding to the barcode sequencefrom the bead (e.g., a spatial barcode) and a DNA sequence correspondingto a target mRNA. Barcoded construct B can include a sequencecorresponding to the barcode sequence from the bead (e.g., a spatialbarcode) and a sequence corresponding to genomic DNA. Barcoded constructC can include a sequence corresponding to the barcode sequence from thebead (e.g., a spatial barcode) and a sequence corresponding to barcodesequence associated with an analyte capture agent (e.g., an analytebinding moiety barcode). Barcoded construct D can include a sequencecorresponding to the barcode sequence from the bead (e.g., a spatialbarcode) and a sequence corresponding to a CRISPR nucleic acid (which,in some embodiments, also includes a barcode sequence). Each constructcan be analyzed (e.g., via any of a variety of sequencing methods) andthe results can be associated with the given cell from which the variousanalytes originated. Barcoded (or even non-barcoded) constructs can betailored for analyses of any given analyte associated with a nucleicacid and capable of binding with such a construct.

In some embodiments of any of the spatial analysis methods describedherein, other combinations of four or more biological analytes that canbe concurrently measured include, without limitation: (a) mRNA, alineage tracing construct, cell surface and/or intracellular proteinsand/or metabolites, and gDNA; (b) mRNA, accessible chromatin (e.g.,ATAC-seq, DNase-seq, and/or MNase-seq), cell surface and/orintracellular proteins and/or metabolites, and a perturbation agent(e.g., a CRISPR crRNA/sgRNA, TALEN, zinc finger nuclease, and/orantisense oligonucleotide as described herein); (c) mRNA, cell surfaceand/or intracellular proteins and/or metabolites, an analyte captureagent (e.g., the MHC multimers described herein), and a V(D)J sequenceof an immune cell receptor (e.g., T-cell receptor); (d) mRNA, genomicDNA, a perturbation reagent, and accessible chromatin; (e) mRNA, cellsurface and/or intracellular proteins and/or metabolites, an analytecapture agent (e.g., the MHC multimers described herein), and aperturbation reagent; (f) mRNA, cell surface and/or intracellularproteins and/or metabolites, a perturbation reagent, and a V(D)Jsequence of an immune cell receptor (e.g., T-cell receptor); (g) mRNA, aperturbation reagent, an analyte capture agent (e.g., the MHC multimersdescribed herein), and a V(D)J sequence of an immune cell receptor(e.g., T-cell receptor); (h) mRNA, chromatin (e.g., spatial organizationof chromatin in a cell), and a perturbation reagent; (i) a V(D)Jsequence of an immune cell receptor, chromatin (e.g., spatialorganization of chromatin in a cell); and a perturbation reagent; (j)mRNA, a V(D)J sequence of an immune cell receptor, chromatin (e.g.,spatial organization of chromatin in a cell), and genomic DNA; (k) mRNA,a V(D)J sequence of an immune cell receptor, chromatin (e.g., spatialorganization of chromatin in a cell), and a perturbation reagent, or anycombination thereof.

(B) Construction of Spatial Arrays for Multi-Analyte Analysis

This disclosure also provides methods and materials for constructing aspatial array capable of multi-analyte analysis. In some embodiments, aspatial array includes a plurality of features on a substrate where oneor more members of the plurality of features include a plurality ofoligonucleotides having a first type functional sequence andoligonucleotides having a second, different type of functional sequence.In some embodiments, a feature can include oligonucleotides with twotypes of functional sequences. A feature can be coupled tooligonucleotides comprising a TruSeq functional sequence and also tooligonucleotides comprising a Nextera functional sequence. In someembodiments, one or more members of the plurality of features comprisesboth types of functional sequences. In some embodiments, one or moremembers of the plurality features includes a first type of functionalsequence. In some embodiments, one or more members of the plurality offeatures includes a second type of functional sequence. In someembodiments, an additional oligonucleotide can be added to thefunctional sequence to generate a full oligonucleotide where the fulloligonucleotide includes a spatial barcode sequence, an optional UMIsequence, a priming sequence, and a capture domain. Attachment of thesesequences can be via ligation (including via splint ligation as isdescribed in U.S. Patent Application Publication No. 20140378345, theentire contents of which are incorporated herein by reference), or anyother suitable route. As discussed herein, oligonucleotides can behybridized with splint sequences that can be helpful in constructingcomplete full oligonucleotides (e.g., oligonucleotides that are capableof spatial analysis).

In some embodiments, the oligonucleotides that hybridize to thefunctional sequences (e.g., TruSeq and Nextera) located on the featuresinclude capture domains capable of capturing different types of analytes(e.g., mRNA, genomic DNA, cell surface proteins, or accessiblechromatin). In some examples, oligonucleotides that can bind to theTruSeq functional sequences can include capture domains that includepoly(T) capture sequences. In addition to the poly(T) capture sequences,the oligonucleotides that can bind the TruSeq functional groups can alsoinclude a capture domain that includes a random N-mer sequence forcapturing genomic DNA (e.g., or any other sequence or domain asdescribed herein capable of capturing any of the biological analytesdescribed herein). In such cases, the spatial arrays can be constructedby applying ratios of TruSeq-poly(T) and TruSeq-N-mer oligonucleotidesto the features comprising the functional TruSeq sequences. This canproduce spatial arrays where a portion of the oligonucleotides cancapture mRNA and a different portion of oligonucleotides can capturegenomic DNA. In some embodiments, one or more members of a plurality offeatures include both TruSeq and Nextera functional sequences. In suchcases, a feature including both types of functional sequences is capableof binding oligonucleotides specific to each functional sequence. Forexample, an oligonucleotide capable of binding to a TruSeq functionalsequence could be used to deliver an oligonucleotide including a poly(T)capture domain and an oligonucleotide capable of binding to a Nexterafunctional sequence could be used to deliver an oligonucleotideincluding an N-mer capture domain for capturing genomic DNA. It will beappreciated by a person of ordinary skill in the art that anycombination of capture domains (e.g., capture domains having any of thevariety of capture sequences described herein capable of binding to anyof the different types of analytes as described herein) could becombined with oligonucleotides capable of binding to TruSeq and Nexterafunctional sequences to construct a spatial array.

In some embodiments, an oligonucleotide that includes a capture domain(e.g., an oligonucleotide capable of coupling to an analyte) or ananalyte capture agent can include an oligonucleotide sequence that iscapable of binding or ligating to an assay primer. The adapter can allowthe capture probe or the analyte capture agent to be attached to anysuitable assay primers and used in any suitable assays. The assay primercan include a priming region and a sequence that is capable of bindingor ligating to the adapter. In some embodiments, the adapter can be anon-specific primer (e.g., a 5′ overhang) and the assay primer caninclude a 3′ overhang that can be ligated to the 5′ overhang. Thepriming region on the assay primer can be any primer described herein,e.g., a poly (T) primer, a random N-mer primer, a target-specificprimer, or an analyte capture agent capture sequence.

In some examples, an oligonucleotide can includes an adapter, e.g., a 5′overhang with 10 nucleotides. The adapter can be ligated to assayprimers, each of which includes a 3′ overhang with 10 nucleotides thatcomplementary to the 5′ overhang of the adapter. The capture probe canbe used in any assay by attaching to the assay primer designed for thatassay.

Adapters and assay primers can be used to allow the capture probe or theanalyte capture agent to be attached to any suitable assay primers andused in any suitable assays. A capture probe that includes a spatialbarcode can be attached to a bead that includes a poly(dT) sequence. Acapture probe including a spatial barcode and a poly(T) sequence can beused to assay multiple biological analytes as generally described herein(e.g., the biological analyte includes a poly(A) sequence or is coupledto or otherwise is associated with an analyte capture agent comprising apoly(A) sequence as the analyte capture sequence).

A splint oligonucleotide with a poly(A) sequence can be used tofacilitate coupling to a capture probe that includes a spatial barcodeand a second sequence that facilitates coupling with an assay primer.Assay primers include a sequence complementary to the splint oligosecond sequence and an assay-specific sequence that determines assayprimer functionality (e.g., a poly(T) primer, a random N-mer primer, atarget-specific primer, or an analyte capture agent capture sequence asdescribed herein).

In some embodiments of any of the spatial profiling methods describedherein, a feature can include a capture probe that includes a spatialbarcode comprising a switch oligonucleotide, e.g., with a 3′ end 3rG.For example, a feature (e.g., a gel bead) with a spatial barcodefunctionalized with a 3rG sequence can be used that enables templateswitching (e.g., reverse transcriptase template switching), but is notspecific for any particular assay. In some embodiments, the assayprimers added to the reaction can determine which type of analytes areanalyzed. For example, the assay primers can include binding domainscapable of binding to target biological analytes (e.g., poly (T) formRNA, N-mer for genomic DNA, etc.). A capture probe (e.g., anoligonucleotide capable of spatial profiling) can be generated by usinga reverse transcriptase enzyme/polymerase to extend, which is followedby template switching onto the barcoded adapter oligonucleotide toincorporate the barcode and other functional sequences. In someembodiments, the assay primers include capture domains capable ofbinding to a poly(T) sequence for mRNA analysis, random primers forgenomic DNA analysis, or a capture sequence that can bind a nucleic acidmolecule coupled to an analyte binding moiety(e.g., a an analyte capturesequence of an analyte capture agent) or a nucleic acid molecule thatcan function in as a perturbation reagent (e.g., a CRISPR crRNA/sgRNA,TALEN, zinc finger nuclease, and/or antisense oligonucleotide asdescribed herein).

V. Systems for Sample Analysis

The methods described above for analyzing biological samples can beimplemented using a variety of hardware components. In this section,examples of such components are described. However, it should beunderstood that in general, the various steps and techniques discussedherein can be performed using a variety of different devices and systemcomponents, not all of which are expressly set forth.

FIG. 46A is a schematic diagram showing an example sample handlingapparatus 4600. Sample handling apparatus 4600 includes a sample chamber4602 that, when closed or sealed, is fluid-tight. Within chamber 4602, afirst holder 4604 holds a first substrate 4606 on which a sample 4608 ispositioned. Sample chamber 4602 also includes a second holder 4610 thatholds a second substrate 4612 with an array of features 4614, asdescribed above.

A fluid reservoir 4616 is connected to the interior volume of samplechamber 4602 via a fluid inlet 4618. Fluid outlet 4620 is also connectedto the interior volume of sample chamber 4602, and to valve 4622. Inturn, valve 4622 is connected to waste reservoir 4624 and, optionally,to analysis apparatus 4626. A control unit 4628 is electricallyconnected to second holder 4610, to valve 4622, to waste reservoir 4624,and to fluid reservoir 4616.

During operation of apparatus 4600, any of the reagents, solutions, andother biochemical components described above can be delivered intosample chamber 4602 from fluid reservoir 4616 via fluid inlet 4618.Control unit 4628, connected to fluid reservoir 4616, can control thedelivery of reagents, solutions, and components, and adjust the volumesand flow rates according to programmed analytical protocols for varioussample types and analysis procedures. In some embodiments, fluidreservoir 4616 includes a pump, which can be controlled by control unit4628, to facilitate delivery of substances into sample chamber 4602.

In certain embodiments, fluid reservoir 4616 includes a plurality ofchambers, each of which is connected to fluid inlet 4618 via a manifold(not shown). Control unit 4628 can selectively deliver substances fromany one or more of the multiple chambers into sample chamber 4602 byadjusting the manifold to ensure that the selected chambers arefluidically connected to fluid inlet 4618.

In general, control unit 4628 can be configured to introduce substancesfrom fluid reservoir 4616 into sample chamber 4602 before, after, orboth before and after, sample 4608 on first substrate 4606 hasinteracted with the array of features 4614 on first substrate 4612. Manyexamples of such substances have been described previously. Examples ofsuch substances include, but are not limited to, permeabilizing agents,buffers, fixatives, staining solutions, washing solutions, and solutionsof various biological reagents (e.g., enzymes, peptides,oligonucleotides, primers).

To initiate interaction between sample 4608 and feature array 4614, thesample and array are brought into spatial proximity. To facilitate thisstep, second holder 4610 - under the control of control unit 4628 - cantranslate second substrate 4612 in any of the x-, y-, and z-coordinatedirections. In particular, control unit 4628 can direct second holder4610 to translate second substrate 4612 in the z-direction so thatsample 4608 contacts, or nearly contacts, feature array 4614.

In some embodiments, apparatus 4600 can optionally include an alignmentsub-system 4630, which can be electrically connected to control unit4628. Alignment sub-system 4630 functions to ensure that sample 4608 andfeature array 4614 are aligned in the x-y plane prior to translatingsecond substrate 4612 in the z-direction so that sample 4608 contacts,or nearly contacts, feature array 4614.

Alignment sub-system 4630 can be implemented in a variety of ways. Insome embodiments, for example, alignment sub-system 4630 includes animaging unit that obtains one or more images showing fiducial markingson first substrate 4606 and/or second substrate 4612. Control unit 4618analyzes the image(s) to determine appropriate translations of secondsubstrate 4612 in the x- and/or y-coordinate directions to ensure thatsample 4608 and feature array 4614 are aligned prior to translation inthe z-coordinate direction.

In certain embodiments, control unit 4628 can optionally regulate theremoval of substances from sample chamber 4602. For example, controlunit 4628 can selectively adjust valve 4622 so that substancesintroduced into sample chamber 4602 from fluid reservoir 4616 aredirected into waste reservoir 4624. In some embodiments, waste reservoir4624 can include a reduced-pressure source (not shown) electricallyconnected to control unit 4628. Control unit 4628 can adjust the fluidpressure in fluid outlet 4620 to control the rate at which fluids areremoved from sample chamber 4602 into waste reservoir 4624.

In some embodiments, analytes from sample 4608 or from feature array4614 can be selectively delivered to analysis apparatus 4626 viasuitable adjustment of valve 4622 by control unit 4628. As describedabove, in some embodiments, analysis apparatus 4626 includes areduced-pressure source (not shown) electrically connected to controlunit 4628, so that control unit 4628 can adjust the rate at whichanalytes are delivered to analysis apparatus 4626. As such, fluid outlet4620 effectively functions as an analyte collector, while analysis ofthe analytes is performed by analysis apparatus 4626. It should be notedthat not all of the workflows and methods described herein areimplemented via analysis apparatus 4626. For example, in someembodiments, analytes that are captured by feature array 4614 remainbound to the array (i.e., are not cleaved from the array), and featurearray 4614 is directly analyzed to identify specifically-bound samplecomponents.

In addition to the components described above, apparatus 4600 canoptionally include other features as well. In some embodiments, forexample, sample chamber 4602 includes a heating sub-system 4632electrically connected to control unit 4628. Control unit 4628 canactivate heating sub-system 4632 to heat sample 4608 and/or featurearray 4614, which can help to facilitate certain steps of the methodsdescribed herein.

In certain embodiments, sample chamber 4602 includes an electrode 4634electrically connected to control unit 4628. Control unit 4628 canoptionally activate electrode 4634, thereby establishing an electricfield between the first and second substrates. Such fields can be used,for example, to facilitate migration of analytes from sample 4608 towardfeature array 4614.

In some of the methods described herein, one or more images of a sampleand/or a feature array are acquired. Imaging apparatus that is used toobtain such images can generally be implemented in a variety of ways.FIG. 46B shows one example of an imaging apparatus 4650. Imagingapparatus 4650 includes a light source 4652, light conditioning optics4654, light delivery optics 4656, light collection optics 4660, lightadjusting optics 4662, and a detection sub-system 4664. Each of theforegoing components can optionally be connected to control unit 4628,or alternatively, to another control unit. For purposes of explanationbelow, it will be assumed that control unit 4628 is connected to thecomponents of imaging apparatus 4650.

During operation of imaging apparatus 4650, light source 4652 generateslight. In general, the light generated by source 4652 can include lightin any one or more of the ultraviolet, visible, and/or infrared regionsof the electromagnetic spectrum. A variety of different light sourceelements can be used to generate the light, including (but not limitedto) light emitting diodes, laser diodes, laser sources, fluorescentsources, incandescent sources, and glow-discharge sources.

The light generated by light source 4652 is received by lightconditioning optics 4654. In general, light conditioning optics 4654modify the light generated by light source 4652 for specific imagingapplications. For example, in some embodiments, light conditioningoptics 4654 modify the spectral properties of the light, e.g., byfiltering out certain wavelengths of the light. For this purpose, lightconditioning optics 4654 can include a variety of spectral opticalelements, such as optical filters, gratings, prisms, and chromatic beamsplitters.

In certain embodiments, light conditioning optics 4654 modify thespatial properties of the light generated by light source 4652. Examplesof components that can be used for this purpose include (but are notlimited to) apertures, phase masks, apodizing elements, and diffusers.

After modification by light conditioning optics 4654, the light isreceived by light delivery optics 4656 and directed onto sample 4608 orfeature array 4614, either of which is positioned on a mount 4658. Lightconditioning optics 4654 generally function to collect and direct lightonto the surface of the sample or array. A variety of different opticalelements can be used for this purpose, and examples of such elementsinclude, but are not limited to, lenses, mirrors, beam splitters, andvarious other elements having non-zero optical power.

Light emerging from sample 4608 or feature array 4614 is collected bylight collection optics 4660. In general, light collection optics 4660can include elements similar to any of those described above inconnection with light delivery optics 4656. The collected light can thenoptionally be modified by light adjusting optics 4662, which cangenerally include any of the elements described above in connection withlight conditioning optics 4654.

The light is then detected by detection sub-system 4664. Generally,detection sub-system 4664 functions to generate one or more images ofsample 4608 or feature array 4614 by detecting light from the sample orfeature array. A variety of different imaging elements can be used indetection sub-system 4664, including CCD detectors and other imagecapture devices.

Each of the foregoing components can optionally be connected to controlunit 4628 as shown in FIG. 46B, so that control unit 4628 can adjustvarious properties of the imaging apparatus. For example, control unit4628 can adjust the position of sample 4608 or feature array 4614relative to the position of the incident light, and also with respect tothe focal plane of the incident light (if the incident light isfocused). Control unit 4628 can also selectively filter both theincident light and the light emerging from the sample.

Imaging apparatus 4650 can typically obtain images in a variety ofdifferent imaging modalities. In some embodiments, for example, theimages are transmitted light images, as shown in FIG. 46B. In certainembodiments, apparatus 4650 is configured to obtain reflection images.In some embodiments, apparatus 4650 can be configured to obtainbirefringence images, fluorescence images, phosphorescence images,multiphoton absorption images, and more generally, any known image type.

In general, control unit 4628 can perform any of the method stepsdescribed herein that do not expressly require user intervention bytransmitting suitable control signals to the components of samplehandling apparatus 4600 and/or imaging apparatus 4650. To perform suchsteps, control unit 4628 generally includes software instructions that,when executed, cause control unit 4628 to undertake specific steps. Insome embodiments, control unit 4628 includes an electronic processor andsoftware instructions that are readable by the electronic processor, andcause the processor to carry out the steps describe herein. In certainembodiments, control unit 4628 includes one or more application-specificintegrated circuits having circuit configurations that effectivelyfunction as software instructions.

Control unit 4628 can be implemented in a variety of ways. FIG. 46C is aschematic diagram showing one example of control unit 4628, including anelectronic processor 4680, a memory unit 4682, a storage device 4684,and an input/output interface 4686. Processor 4680 is capable ofprocessing instructions stored in memory unit 4682 or in storage device4684, and to display information on input/output interface 4686.

Memory unit 4682 stores information. In some embodiments, memory unit4682 is a computer-readable medium. Memory unit 4682 can includevolatile memory and/or non-volatile memory. Storage device 4684 iscapable of providing mass storage, and in some embodiments, is acomputer-readable medium. In certain embodiments, storage device 4684may be a floppy disk device, a hard disk device, an optical disk device,a tape device, a solid state device, or another type of writeablemedium.

The input/output interface 4686 implements input/output operations. Insome embodiments, the input/output interface 4686 includes a keyboardand/or pointing device. In some embodiments, the input/output interface4686 includes a display unit for displaying graphical user interfacesand/or display information.

Instructions that are executed and cause control unit 4628 to performany of the steps or procedures described herein can be implemented indigital electronic circuitry, or in computer hardware, firmware, or incombinations of these. The instructions can be implemented in a computerprogram product tangibly embodied in an information carrier, e.g., in amachine-readable storage device, for execution by a programmableprocessor (e.g., processor 4680). The computer program can be written inany form of programming language, including compiled or interpretedlanguages, and it can be deployed in any form, including as astand-alone program or as a module, component, subroutine, or other unitsuitable for use in a computing environment. Storage devices suitablefor tangibly embodying computer program instructions and data includeall forms of non-volatile memory, including by way of examplesemiconductor memory devices, such as EPROM, EEPROM, and flash memorydevices; magnetic disks such as internal hard disks and removable disks;magneto-optical disks; and CD-ROM and DVD-ROM disks. The processor andthe memory can be supplemented by, or incorporated in, ASICs(application-specific integrated circuits).

Processor 4680 can include any one or more of a variety of suitableprocessors. Suitable processors for the execution of a program ofinstructions include, by way of example, both general and specialpurpose microprocessors, and the sole processor or one of multipleprocessors of any kind of computer or computing device.

VI. Systems, Methods, and Compositions for Method forTransposase-Mediated Spatial Tagging and Analyzing Genomic DNA in aBiological Sample

The human body includes a large collection of diverse cell types, eachproviding a specialized and context-specific function. Understanding acell’s chromatin structure can reveal information about the cell’sfunction. Open chromatin, or accessible chromatin, is often indicativeof transcriptionally active sequences, e.g., genes, in a particularcell. Further understanding the transcriptionally active regions withinchromatin will enable identification of which genes contribute to acell’s function and/or phenotype.

Methods have been developed to study epigenomes, e.g., chromatinaccessibility assays (ATAC-seq) or identifying proteins associated withchromatin e.g., (ChIP-seq). These assays help identify regulators (e.g.,cis regulators and/or trans regulators) that contribute to dynamiccellular phenotypes. While ATAC-Seq and ChIP-Seq have been invaluable indefining epigenetic variability within a cell population, conventionalapplications of these methods are limited in their ability to spatiallyresolve the three dimensional structures and associated genes thatpromote cellular variation.

Thus, the present disclosure relates generally to the spatial taggingand analysis of nucleic acids. In some embodiments, provided herein aremethods that utilize a transposase enzyme to facilitate the capture offragmented DNA and enable the simultaneous capture of DNA and RNA from abiological sample, thus revealing epigenomic insights regarding thestructural features contributing to cellular regulation.

In some embodiments, provided herein are methods for spatial analysis ofnucleic acids (e.g., genomic DNA, mRNA) in a biological sample. In someembodiments, a substrate is provided, wherein the substrate comprises aplurality of capture probes. In some embodiments, the capture probes maybe attached directly to the substrate. In some embodiments, the captureprobes may be attached indirectly to the substrate. For example, thecapture probes can be attached to features on the substrate. In someembodiments, the capture probes comprise a spatial barcode and a capturedomain. In some embodiments, the capture probe can be partially doublestranded. In some embodiments, the capture probe can bind acomplementary oligonucleotide. In some embodiments, the complementaryoligonucleotide (e.g., splint oligonucleotide) can have a singlestranded capture domain. In some embodiments, the single strandedcapture domain can bind fragmented (e.g., tagmented) DNA. In someembodiments, the complementary oligonucleotide with the single strandedcapture domain can be a splint oligonucleotide. In some embodiments, abiological sample is treated under conditions sufficient to make nucleicacids in cells of the biological sample (e.g., genomic DNA) accessibleto a transposon insertion. In some embodiments, a transposon sequenceand a transposase enzyme are provided to the biological sample such thatthe transposon sequence can be inserted into the genomic DNA of cellspresent in the biological sample. In some embodiments, the transposaseenzyme can excise (e.g., cut out, remove) the inserted transposonsequence from the nucleic acid (e.g., genomic DNA), thereby fragmentingthe genomic DNA.

In some embodiments, the biological sample comprising nucleic acids(e.g., genomic DNA, mRNA) is contacted to the substrate such that acapture probe can interact with the fragmented (e.g., tagmented) genomicDNA. In some embodiments, the biological sample comprising nucleic acids(e.g., genomic DNA, mRNA) is contacted with the substrate such that thecapture probe can interact with both the fragmented genomic DNA and themRNA present in the biological sample.

In some embodiments, the location of the capture probe on the substratecan be correlated to a location in the biological sample, therebyspatially analyzing the fragmented (e.g., tagmented) genomic DNA. Insome embodiments, the location of the capture probe on the substrate canbe correlated to a location in the biological sample, thereby spatiallyanalyzing the fragmented genomic DNA and mRNA.

Spatial ATAC-seq

In some embodiments, of any of the spatial analysis methods describedherein, ATAC-seq is used to generate genome-wide chromatin accessibilitymaps. These genome-wide accessibility maps can be integrated withadditional genome-wide profiling data (e.g., RNA-seq, ChIP-seq,Methyl-Seq) to produce gene regulatory interaction maps that facilitateunderstanding of transcriptional regulation. For example, interrogationof genome-wide accessibility maps can reveal the underlyingtranscription factors and the transcription factor motifs responsiblefor chromatin accessibility at a given genomic location. Correlatingchanges in chromatin accessibility with changes in gene expression(RNA-seq), changes in TF binding (e.g., ChIP-seq) and/or changes in DNAmethylation levels (e.g., Methyl-seq) can identify the transcriptionregulation driving these changes. In disease states, there is often animbalance in this transcriptional regulation. Thus, analyzing bothchromatin accessibility and, for example, gene expression using spatialanalysis methods enables identification of causes underlying theimbalances in transcriptional regulation.

In some embodiments, where spatial profiling includes concurrentanalysis of different types of analytes from a single cell or asubpopulation of cells within a biological sample (e.g., a tissuesection), an additional layer of spatial information can be integratedinto the genome regulatory interaction maps. In some embodiments, thespatial profiling can be done on whole genomes. In some embodiments, thespatial profiling can be done on an immobilized biological sample (e.g.,fixed biological sample).

In some embodiments, the genome-wide chromatin accessibility mapsgenerated by spatial ATAC-seq can be used for cell type identification.For example, traditional cell type classification relies on mRNAexpression levels but chromatin accessibility can be more adept atcapturing cell identity. Furthermore, in some embodiments, correlationsbetween transcriptionally active regions (e.g., open chromatin) withexpression profiles (e.g., expression profiles of mRNA) can bedetermined in a spatial manner.

Permeabilizing the Biological Sample

The present disclosure generally describes methods of fragmenting (e.g.,tagmenting) genomic DNA to generate DNA fragments in a biologicalsample. Generally, a biological sample needs to be permeabilized underconditions sufficient to access genomic DNA. However, permeabilizationconditions typically used in DNA tagmentation reactions in cellularpreparations (e.g., IGEPAL, Digitonin, NP-40, Tween or Triton-X-100) areinsufficient to enable successful fragmentation (e.g., tagmentation) inbiological samples immobilized on a substrate, e.g., a support, anarray. As described further in the Examples below, a chemical orenzymatic “pre-permeabilization” of biological samples immobilized on asubstrate can be employed to make DNA in the biological sampleaccessible to a transposase enzyme (e.g. a transposome). In someembodiments, permeabilizing the biological sample can be a two-stepprocess (e.g., pre-permeabilization treatment, followed by apermeabilization treatment). In some embodiments, permeabilizing thebiological sample can be a one-step process (e.g., a singlepermeabilization treatment sufficient to permeabilize the cellular andnuclear membranes in the biological sample). In some embodiments, the“pre-permeabilization” conditions can be adapted to yield uniform DNAfragmentation to enable capture of DNA tagments regardless of chromatinaccessibility or to yield fragments with a pronounced nucleosomalpattern.

In some embodiments, pre-permeabilization can include an enzymatic orchemical condition. In some embodiments, pre-permeabilization can beperformed with an enzyme (e.g., a protease). In some embodiments, in anon-limiting way, the protease can include trypsin, pepsin, dispase,papain, accuses, or collagenase. In some embodiments,pre-permeabilization can include an enzymatic treatment with pepsin. Insome embodiments, pre-permeabilization can include pepsin in 0.5 Macetic acid. In some embodiments, pre-permeabilization can includepepsin in Exonuclease-1 buffer. In some embodiments, the pH of thebuffer can be acidic. In some embodiments, pre-permeabilization caninclude enzymatic treatment with collagenase. In some embodiments,pre-permeabilization can include collagenase in HBSS buffer. In someembodiments, the HBSS buffer can include bovine serum albumin (BSA). Insome embodiments, pre-permeabilization can include Proteinase K in PKDbuffer. In some embodiments, the ratio of Proteinase K to PKD Buffer canbe between about 1:1 to about 1:20. In some embodiments, the ratio ofProteinase K to PKD Buffer can be between about 1:5 to about 1:15. Insome embodiments, the ratio of Proteinase K to PKD Buffer can be about1:8. In some embodiments, enzymatic treatment with Proteinase K can beat about 37° C. In some embodiments, pre-permeabilization can include anenzymatic treatment with trypsin. In some embodiments, enzymatictreatment with trypsin can be at about 20° C., about 30° C., or about40° C. In some embodiments, enzymatic treatment with trypsin can be atabout 37° C. In some embodiments, pre-permeabilization can last forabout 1 to minute to about 20 minutes. In some embodiments,pre-permeabilization can last for about 2, about 3, about 4, about 5,about 6, about 7, about 8, about 9, about 10, about 11, about 12, about13, about 14, about 15, about 16, about 17, about 18, or about 19minutes. In some embodiments, pre-permeabilization can last for about 10minutes to about one hour. For example, in some embodiments,pre-permeabilization can last for about 20, about 30, about 40, or about50 minutes.

In some embodiments, permeabilizing the biological sample comprises anenzymatic treatment. In some embodiments, the enzymatic treatment can bea pepsin enzyme, or a pepsin-like enzyme treatment. In some embodiments,the enzymatic treatment can be protease treatment. In some embodiments,enzymatic treatment can be performed in the presence of reagents. Insome embodiments, the enzymatic treatment (e.g., pre-permeabilization)can include contacting the biological specimen with an acidic solutionincluding a protease enzyme. In some embodiments, the reagent can beHCl. In some embodiments, the reagent can be acetic acid. In someembodiments, the concentration of HCl can be about 100 mM. In someembodiments, the about 100 mM HCl can have a pH of around, or about 1.0.In some embodiments, the additional reagent can be 0.5 M acetic acid,having a pH of around, or about 2.5. It is noted that enzymatictreatment of the biological sample can have different effects ontagmentation. For example, enzymatic treatment with pepsin and 100 mMHCl can result in tagmentation of chromatin regardless of chromatinaccessibility. In some embodiments, enzymatic treatment with pepsin and0.5 M acetic acid can result in tagmentation of chromatin that canretain a nucleosomal pattern indicative of tagmentation.

In some embodiments, the enzymatic treatment can comprise contacting thebiological sample with a reaction mixture (e.g., solution) comprising anaspartyl protease (e.g., pepsin) in an acidic buffer, e.g., a bufferwith a pH of about 4.0 or less, such as about 3.0 or less, e.g., about0.5 to about 3.0, or about 1.0 to about 2.5. In some embodiments, theaspartyl protease is a pepsin enzyme, pepsin-like enzyme, or afunctional equivalent thereof. Thus, any enzyme or combination ofenzymes in the enzyme commission number 3.4.23.1.

In some embodiments, the enzymatic treatment with pepsin enzyme, orpepsin like enzyme, can be selected from the following(UniProtKB/Swiss-Prot accession numbers): P03954/PEPA1_MACFU;P28712/PEPA1_RABIT; P27677/PEPA2_MACFU; P27821/PEPA2_RABIT;P0DJD8/PEPA3_HUMAN; P27822/PEPA3_RABIT; P0DJD7/PEPA4_(_)HUMAN;P27678/PEPA4_MACFU; P28713/PEPA4_RABIT; P0DJD9/PEPA5_(_)HUMAN;Q9D106/PEPA5_MOUSE; P27823/PEPAF_RABIT; P00792/PEPA_BOVIN;Q9N2D4/PEPA_CALJA; Q9GMY6/PEPA_CANLF; P00793/PEPA_CHICK;P11489/PEPA_MACMU; P00791/PEPA_PIG; Q9GMY7/PEPA_RHIFE;Q9GMY8/PEPA_SORUN; P81497/PEPA_SUNMU; P13636/PEPA_URSTH and functionalvariants and derivatives thereof, or a combination thereof. In someembodiments, the pepsin enzyme is selected from (UniProtKB/Swiss-Protaccession numbers): P00791/PEPA_PIG; P00792/PEPA_BOVIN and functionalvariants and derivatives thereof or a combination thereof.

In some embodiments, the pepsin enzyme or functional variant orderivative thereof, comprises an amino acid sequence with at least 80%sequence identity to a sequence as set forth in SEQ ID NOs: 3 or 4.Preferably the polypeptide includes a sequence having at least about 90,91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to the sequenceto which it is compared (e.g., SEQ ID NOs. 3 or 4).

In some embodiments, the enzymatic treatment (e.g.,pre-permeabilization) can be a Proteinase K or Proteinase K-liketreatment. In some embodiments, enzymatic treatment with Proteinase Kcan result in tagmentation of accessible chromatin in the biologicalsample. In some embodiments, enzymatic treatment (e.g.,pre-permeabilization and permeabilization) with Proteinase K can resultin tagmentation of inaccessible chromatin, (e.g., nucleosomal DNA). Insome embodiments, the enzymatic treatment comprises contacting thebiological sample with a serine protease (e.g., Proteinase K) withreagents and under conditions suitable for proteolytic activity. Forexample, the serine protease is functional under a wide range of pHconditions (e.g., from about 6.5 to about 9.5), denaturing conditions(e.g., presence of SDS, urea), metal chelating agents (e.g., EDTA), andtemperatures (e.g., about 45° to about 65°). In some embodiments, it canbe useful to stop enzymatic activity of the serine protease (e.g.,Proteinase K) with an inhibitor. For example, following enzymatictreatment, Proteinase K can be inhibited by a small molecule (e.g.,Sigma Cat. No. 539470).

In some embodiments, the serine protease is a proteinase K enzyme,proteinase K-like enzyme, or a functional equivalent thereof. Forexample, any enzyme or combination of enzymes in the enzyme commissionnumber 3.4.21.64 can be used. In some embodiments, the Proteinase K isP06873/PRTK_PARAQ, (UniProtKB/Swiss-Prot accession number), or afunctional variant or derivative thereof (as described herein), or acombination thereof.

In some embodiments, the proteinase K enzyme, or functional variant orderivative thereof, comprises an amino acid sequence with at least 80%sequence identity to a sequence as set forth in SEQ ID NO. 7. In someembodiments, the polypeptide sequence is an amino acid sequence havingabout at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequenceidentity to the sequence to which it is compared (e.g. SEQ ID NO. 7)

In some embodiments, the enzymatic treatment (e.g.,pre-permeabilization) can be performed using collagenase. In someembodiments, enzymatic treatment with collagenase can provide access tothe genomic DNA for the transposase while preserving nuclear integrity.In some embodiments, pre-permeabilization (e.g., enzymatic treatment)with collagenase yields nucleosomal patterns generally associated withtagmentation. Collagenases can be isolated from Clostridiumhistolyticum. In some embodiments, enzymatic treatment with a zincendopeptidase (e.g., collagenase) with reagents and under conditionssuitable for proteolytic activity comprises a buffered solution with apH of about 7.0 to about 8.0 (e.g., about 7.4). Collagenases are zincendopeptidases and can be inhibited by either EDTA or EGTA, or both.Therefore, in some embodiments, the biological sample can be contactedwith a zinc endopeptidase (e.g., collagenase) in the absence of achelator of divalent cations, (e.g., EDTA, EGTA). In some embodiments,it can be useful to stop the zinc endopeptidase (e.g., collagenase) andthe permeabilization step can be stopped (e.g., inhibited) by contactingthe biological sample with a chelator of divalent cations (e.g., EDTA,EGTA).

In some embodiments, the zinc endopeptidase is a collagenase enzyme,collagenase-like enzyme, or a functional equivalent thereof. In suchembodiments, any enzyme or combination of enzymes in the enzymecommission number 3.4.23.3 can be used in accordance with materials andmethods described herein. In some embodiments, the collagenase is one ormore collagenases from the following group, (UniProtKB/Swiss-Protaccession numbers): P43153/COLA_CLOPE; P43154/COLA_VIBAL;Q9KRJ0/COLA_VIBCH; Q56696/COLA_VIBPA; Q8D4Y9/COLA_VIBVU;Q9X721/COLG_HATHI; Q46085/COLH_HATHI; Q899Y1/COLT_CLOTE URSTH andfunctional variants and derivatives thereof (described herein), or acombination thereof.

In some embodiments, the collagenase enzyme, or functional variant orderivative thereof, comprises an amino acid sequence with at least 80%sequence identity to a sequence as set forth in SEQ ID NOs. 5 or 6. Insome embodiments, said polypeptide sequence is a sequence having atleast about 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identityto the sequence to which it is compared (e.g., SEQ ID NOs. 5 or 6).

Methods of permeabilizing biological samples are well known in the art.It will be known to a person skilled in the art that different sourcesof biological samples can be treated with different reagents (e.g.,proteases, RNAses, detergents, buffers) and under different conditions(e.g., pressure, temperature, concentration, pH, time). In someembodiments, permeabilizing the biological sample can comprise reagentsand conditions to sufficiently disrupt the cell membrane of thebiological sample to capture nucleic acid (e.g., mRNA). In someembodiments, permeabilizing the biological sample can comprise reagentsand conditions to sufficiently disrupt the nuclear membrane of thebiological sample to capture nucleic acid (e.g., genomic DNA). In someembodiments, commercially available proteases isolated from their native(e.g., animal, microbial source) can be used. In some embodiments,proteases produced recombinantly (e.g., bacterial expression system) canbe used. In some embodiments, pre-permeabilizing and permeabilizing abiological sample can be a one-step process (e.g., enzymatic treatment).In some embodiments, pre-permeabilizing and permeabilizing a biologicalsample can be a two-step process (e.g., enzymatic treatment, followed bychemical or detergent treatment).

In some embodiments, the chemical permeabilization conditions comprisecontacting the biological specimen with an alkaline solution, e.g. abuffered solution with a pH of about 8.0 to about 11.0, such as about8.5 to about 10.5 or about 9.0 to about 10.0, e.g. about 9.5. In someembodiments, the buffer is a glycine-KOH buffer. Other buffers are knownin the art.

In some embodiments, a biological sample can be treated with a detergentfollowing an enzymatic treatment (e.g., permeabilization following apre-permeabilization step). Detergents are known in the art. Anysuitable detergent can be used, including, in a non-limiting way NP-40or equivalent, Digitonin, Tween-20, IGEPAL-40 or equivalent, Saponin,SDS, Pitsop2, or combinations thereof. In some embodiments, a biologicalsample can be treated with other chemicals known to permeabilizecellular membranes. As further exemplified in the examples below,detergents described herein can be used at a concentration of betweenabout 0.01% to about 0.1%. In some embodiments, detergents describedherein can be used at a concentration of about 0.2%, about 0.3%, about0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, or about 0.9%. Insome embodiments, detergents described herein can be used at aconcentration of about 1.1% to about 10% or more. In some embodiments,detergents described herein can be used at a concentration of about 2%,about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, or about 9%.

Additional methods for sample permeabilization are described, forexample, in Jamur et al., Method Mol. Biol. 588:63-66, 2010, the entirecontents of which are incorporated herein by reference. Any suitablemethod for biological sample permeabilization can generally be used inconnection with the biological samples described herein.

Different sources of biological samples can be treated with differentreagents (e.g., proteases, RNAses, detergents, buffers) and underdifferent suitable conditions (e.g., pressure, temperature,concentration, pH, time) to achieve sufficient pre-permeabilization andpermeabilization to capture nucleic acids (e.g., genomic DNA, mRNA). Forexample, enzymatic treatment (e.g., pepsin, collagenase, Proteinase K)can be used at a concentration of about 0.05 mg/ml to about 1 mg/ml,e.g., about 0.1 mg/ml to about 0.5 mg/ml. In some embodiments, enzymatictreatment can be used at a concentration of about 1.1 mg/ml to about 1.9mg/mL. In some embodiments, the biological sample can be incubated withthe protease enzymes and/or chemical reagents (e.g., alkaline buffer)for about 1-10 minutes, e.g., about 2, about 3, about 4, about 5, about6, about 7, about 8, or about 9 minutes. In some embodiments, thebiological sample can be incubated with the protease enzymes and/orchemical reagents (e.g., alkaline buffer) for at least about 5 minutes,e.g. at least about 10, about 12, about 15, about 18, or about 20minutes. For instance, the collagenase enzymes (or functionalequivalents thereof) can be incubated with the biological sample forabout 10 to about 30 minutes, e.g., about 20 minutes.

In some embodiments, the biological sample can be incubated with theprotease enzymes and/or chemical reagents (e.g., alkaline buffer) for upto about 1 hour, e.g., for up to about 10, about 20, about 30, about 40,or about 50 minutes. In some embodiments, the biological sample can beincubated with the protease enzymes and/or chemical reagents (e.g.,alkaline buffer) for up to about 4 hours, e.g., for up to about 2 orabout 3 hours. The incubation period can depend on the concentration ofthe enzyme and the conditions of use, e.g., buffer, temperature etc. Insome embodiments, the protease enzymes can be incubated with thebiological specimen for more or less time than the periods set outabove.

In some embodiments, pre-permeabilization and permeabilizationconditions can be impacted by various temperatures. For example,representative temperature conditions for the pre-permeabilization andpermeabilization step include incubation at about 10 to about 70° C.,depending on the enzyme. For example, pepsin and collagenase may be usedat about 10 to about 44°, about 11 to about 43°, about 12 to about 42°,about 13 to about 41°, about 14 to about 40°, about 15 to about 39°,about 16 to about 38°, about 17 to about 37° C., e.g., about 10°, about12°, about 15°, about 18°, about 20°, about 22°, about 25°, about 28°,about 30°, about 33°, about 35°, or about 37° C., e.g., about 30 toabout 40° C., e.g., about 37° C. Proteinase K may be used at about 40 toabout 70° C., e.g. about 50to about 70° C., about 60 to about 70° C.e.g., about 65° C.

In some embodiments, the pre-permeabilization and permeabilization stepcan be stopped (e.g., the protease activity may be stopped) by anysuitable means. For instance, the reaction mixture (e.g., solution)comprising the protease enzymes and/or chemical reagents can be removedfrom the substrate (e.g., a support) and separated from the biologicalsample. Alternatively or additionally, the protease enzyme(s) can beinhibited (e.g., by the addition of an inhibitor, such as EDTA forcollagenase) or denatured (e.g., by the addition of a denaturing agentor increasing the temperature).

In some embodiments, the reaction mixture (e.g., solution) including theproteases described herein can contain other reagents, (e.g., buffer,salt, etc.) sufficient to ensure that the proteases are functional. Forinstance, the reaction mixture can further include an albumin protein,(e.g., BSA). In some embodiments, the reaction mixture (e.g., solution)including the collagenase enzyme (or functional variant or derivativethereof) includes an albumin protein, (e.g., BSA).

Tagmentation

Transposase enzymes and transposons can be utilized in methods ofspatial genomic analysis. Generally, transposition is the process bywhich a specific genetic sequence (e.g., a transposon sequence) isrelocated from one place in a genome to another. Many transpositionmethods and transposable elements are known in the art (e.g., DNAtransposons, retrotransposons, autonomous transposons, non-autonomoustransposons). One non-limiting example of a transposition event isconservative transposition. Conservative transposition is anon-replicative mode of transposition in which the transposon iscompletely removed from the genome and reintegrated into a new locus,such that the transposon sequence is conserved, (e.g., a conservativetransposition event can be thought of as a “cut and paste” event) (See,e.g., Griffiths A. J., et. al., Mechanism of transposition inprokaryotes. An Introduction to Genetic Analysis (7th Ed.). New York: W.H. Freeman (2000)).

In one example, cut and paste transposition can occur when a transposaseenzyme binds a sequence flanking the ends of the transposome (e.g., arecognition sequence, e.g., a mosaic end sequence). A transposome (e.g.,a transposition complex) forms and the endogenous DNA can be manipulatedinto a pre-excision complex such that two transposases enzymes caninteract. In some embodiments, when the transposases interact doublestranded breaks are introduced into the DNA resulting in the excision ofthe transposon sequence. The transposase enzymes can locate and bind atarget site in the DNA, create a double stranded break, and insert thetransposon sequence (See, e.g., Skipper, K.A., et. al., DNAtransposon-based gene vehicles-scenes from an evolutionary drive, JBiomed Sci., 20: 92 (2013) doi:10.1186/1423-0127-20-92). Alternative cutand paste transposases include Tn552 (College, et al, J. BacterioL, 183:2384-8, 2001; Kirby C et al, Mol. Microbiol, 43: 173-86, 2002), Tyl(Devine & Boeke, Nucleic Acids Res., 22: 3765-72, 1994 and InternationalPublication WO 95/23875), Transposon Tn7 (Craig, N L, Science. 271 :1512, 1996; Craig, N L, Review in: Curr Top Microbiol Immunol,204:27-48, 1996), Tn/O and IS10 (Kleckner N, et al, Curr Top MicrobiolImmunol, 204:49-82, 1996), Mariner transposase (Lampe D J, et al, EMBOJ., 15: 5470-9, 1996), Tel (Plasterk R H, Curr. Topics Microbiol.Immunol, 204: 125-43, 1996), P Element (Gloor, G B, Methods Mol. Biol,260: 97- 114, 2004), Tn3 (Ichikawa & Ohtsubo, J Biol. Chem. 265:18829-32, 1990), bacterial insertion sequences (Ohtsubo & Sekine, Curr.Top. Microbiol. Immunol. 204: 1-26, 1996), retroviruses (Brown, et al,Proc Natl Acad Sci USA, 86:2525-9, 1989), and retrotransposon of yeast(Boeke & Corces, Annu Rev Microbiol. 43:403-34, 1989). More examplesinclude IS5, TnlO, Tn903, IS911, and engineered versions of transposasefamily enzymes (Zhang et al, (2009) PLoS Genet. 5:el000689. Epub 2009Oct. 16; Wilson C. et al (2007) J. Microbiol. Methods 71 :332-5).

In some methods of spatial genomic analysis, DNA is fragmented in such amanner that a sequence complementary to a capture domain of a captureprobe (e.g., capture domain of a splint oligonucleotide) is attached tothe fragmented DNA (e.g., the fragmented DNA is “tagged”), such that theattached sequence (e.g. an adapter, e.g., Nextera adapter) can hybridizeto the capture probe. In some embodiments, the capture probe is presenton a substrate. In some embodiments, the capture probe (e.g., a surfaceprobe and a splint oligonucleotide) is present on a feature.Transposome-mediated fragmentation (“tagmentation”) is a process oftransposase-mediated fragmentation and tagging of DNA. A transposome isa complex of a transposase enzyme and DNA which comprises a transposonend sequence (also known as “transposase recognition sequence” or“mosaic end” (MEs)). A transposome dimer is able to simultaneouslyfragment DNA based on its transposon recognition sequences and ligateDNA from the transposome to the fragmented DNA (e.g., tagmented DNA).This system has been adapted using hyperactive transposase enzymes andmodified DNA molecules (adaptors) comprising MEs to fragment DNA and tagboth strands of DNA duplex fragments with functional DNA molecules(e.g., primer binding sites). For instance, the Tn5 transposase may beproduced as purified protein monomers. Tn5 transposase is alsocommercially available (e.g., manufacturer Illumina, Illumina.com,Catalog No. 15027865, TD Tagment DNA Buffer Catalog No. 15027866). Thesecan be subsequently loaded with the oligonucleotides of interest, e.g.,ssDNA oligonucleotides containing MEs for Tn5 recognition and additionalfunctional sequences (e.g., Nextera adapters, e.g., primer bindingsites) are annealed to form a dsDNA mosaic end oligonucleotide (MEDS)that is recognized by Tn5 during dimer assembly (e.g., transposomedimerization). In some embodiments, a hyperactive Tn5 transposase can beloaded with adapters (e.g., oligonucleotides of interest) which cansimultaneously fragment and tag a genome with the adapter sequences.

As used herein, the term “tagmentation” refers to a step in the Assayfor Transposase Accessible Chromatin using sequencing (ATAC-seq). (See,e.g., Buenrostro, J. D., Giresi, P. G., Zaba, L. C, Chang, H. Y.,Greenleaf, W. J., Transposition of native chromatin for fast andsensitive epi genomic profiling of open chromatin, DNA-binding proteinsand nucleosome position, Nature Methods, 10 (12): 1213-1218 (2013)).ATAC-seq identifies regions of open chromatin using a hyperactiveprokaryotic Tn5-transposase, which preferentially inserts intoaccessible chromatin and tags the sites with adaptors (Buenrostro, J.D.,et. al., Transposition of native chromatin for fast and sensitiveepigenomic profiling of open chromatin, DNA-binding proteins andnucleosome position. Nat Methods, 10: 1213-1218 (2013)).

In some embodiments, the step of fragmenting the genomic DNA in cells ofthe biological sample comprises contacting the biological samplecontaining the genomic DNA with the transposase enzyme (e.g., atransposome, e.g., a reaction mixture (e.g., solution)) including atransposase), under any suitable conditions. In some embodiments, suchsuitable conditions result in the fragmentation (e.g., tagmentation) ofthe genomic DNA of cells present in the biological sample. Typicalconditions will depend on the transposase enzyme used and can bedetermined using routine methods known in the art. Therefore, suitableconditions can be conditions (e.g., buffer, salt, concentration, pH,temperature, time conditions) under which the transposase enzyme isfunctional, e.g., in which the transposase enzyme displays transposaseactivity, particularly tagmentation activity, in the biological sample.

The term “functional”, as used herein in reference to transposaseenzymes, is meant to include embodiments in which the transposase enzymecan show some reduced activity relative to the activity of thetransposase enzyme in conditions that are optimum for the enzyme, e.g.,in the buffer, salt and temperature conditions recommended by themanufacturer. Thus, the transposase can be considered to be “functional”if it has at least about 50%, e.g., at least about 60, about 70, about80, about 85, about 90, about 95, about 96, about 97, about 98, about99, or about 100%, activity relative to the activity of the transposasein conditions that are optimum for the transposase enzyme.

In one non-limiting example, the reaction mixture comprises atransposase enzyme in a buffered solution (e.g., Tris-acetate) having apH of about 6.5 to about 8.5, e.g., about 7.0 to about 8.0 such as about7.5. Additionally or alternatively, the reaction mixture can be used atany suitable temperature, such as about 10° to about 55° C., e.g., about10° to about 54°, about 11° to about 53°, about 12° to about 52°, about13° to about 51°, about 14° to about 50°, about 15° to about 49°, about16° to about 48°, about 17° to about 47° C., e.g., about 10°, about 12°,about 15°, about 18°, about 20°, about 22°, about 25°, about 28°, about30°, about 33°, about 35°, about or 37° C., preferably about 30° toabout 40° C., e.g., about 37° C. In some embodiments, the transposaseenzyme can be contacted with the biological sample for about 10 minutesto about one hour. In some embodiments, the transposase enzyme can becontacted with the biological sample for about 20, about 30, about 40,or about 50 minutes. In some embodiments, the transposase enzyme can becontacted with the biological sample for about 1 hour to about 4 hours.

In some embodiments, the transposase enzyme is a Tn5 transposase, or afunctional derivate or variant thereof. (See, e.g., Reznikoff et al, WO2001/009363, U.S. Pat. Nos. 5,925,545, 5,965,443, 7,083,980, and7,608,434, and Goryshin and Reznikoff, J. Biol. Chem. 273:7367, (1998),which are herein incorporated by reference). For example, the Tn5transposase can be a fusion protein (e.g., a Tn5 fusion protein). Tn5 isa member of the RNase superfamily of proteins. The Tn5 transposon is acomposite transposon in which two near-identical insertion sequences(IS50L and IS50R) flank three antibiotic resistance genes. Each IS50contains two inverted 19-bp end sequences (ESs), an outside end (OE) andan inside end (IE). Wild-type Tn5 transposase enzyme is generallyinactive (e.g., low transposition event activity). However, amino acidsubstitutions can result in hyperactive variants or derivatives. In onenon-limiting example, amino acid substitution, L372P, substitutes aleucine amino acid for a proline amino acid which results in an alphahelix break, thus inducing a conformational change to the C-terminaldomain. The alpha helix break separates the C-terminal domain andN-terminal domain sufficiently to promote higher transposition eventactivity (See, Reznikoff, W.S., Tn5 as a model for understanding DNAtransposition, Mol Microbiol, 47(5): 1199-1206 (2003)). Other amino acidsubstitutions resulting in hyperactive Tn5 are known in the art. Forexample, the improved avidity of the modified transposase enzyme (e.g.,modified Tn5 transposase enzyme) for the repeat sequences for OE termini(class (1) mutation) can be achieved by providing a lysine residue atamino acid 54, which is glutamic acid in wild-type Tn5 transposaseenzyme (See U.S. Pat. No. 5.925,545). The mutation strongly alters thepreference of the modified transposase enzyme (e.g., modified Tn5transposase enzyme) for OE termini, as opposed to IE termini. The higherbinding of this mutation, known as EK54, to OE termini results in atransposition rate that is about 10-fold higher than is seen withwild-type transposase enzyme (e.g., wild type Tn5 transposase enzyme). Asimilar change at position 54 to valine (e.g., EV54) also results insomewhat increased binding/transposition for OE termini, as does athreonine to proline change at position 47 (e.g., TP47; about 10-foldhigher) (See U.S. Pat. No. 5.925,545).

Other examples of modified transposase enzymes (e.g., modified Tn5transposase enzymes) are known. For example, a modified Tn5 transposaseenzyme that differs from wild-type Tn5 transposase enzyme in that itbinds to the repeat sequences of the donor DNA with greater avidity thanwild-type Tn5 transposase enzyme and also is less likely than thewild-type transposase enzyme to assume an inactive multimeric form (U.S.Pat. No. 5,925,545, which is incorporated by reference in its entirety).Furthermore, techniques generally describing introducing anytransposable element (e.g., Tn5) from a donor DNA (e.g., adaptersequence, e.g., Nextera adapters (e.g., top and bottom adapter) into atarget are known in the art. (See, e.g., U.S. Pat. No. 5,925,545).Further study has identified classes of mutations resulting in amodified transposase enzyme (e.g., modified Tn5 transposase enzyme)(See, U.S. Pat. No. 5,965,443, which is incorporated by reference in itsentirety). For example, a modified transposase enzyme (e.g., modifiedTn5 transposase enzyme) with a “class 1 mutation” binds to repeatsequences of donor DNA with greater avidity than wild-type Tn5transposase enzyme. Additionally, a modified transposase enzyme (e.g.,modified Tn5 transposase enzyme) with a “class 2 mutation” is lesslikely than the wild-type Tn5 transposase enzyme to assume an inactivemultimeric form. It has been shown that a modified transposase enzymethat contains both a class 1 and a class 2 mutation can induce at leastabout 100-fold (+10%) more transposition than the wild-type transposaseenzyme, when tested in combination with an in vivo conjugation assay asdescribed by Weinreich, M.D., “Evidence that the cis Preference of theTn5 Transposase is Caused by Nonproductive Multimerization,” Genes andDevelopment 8:2363-2374 (1994), incorporated herein by reference (Seee.g., U.S. Pat. No. 5,965,443). Further, under sufficient conditions,transposition using the modified transposase enzyme (e.g., modified Tn5transposase enzyme) may be higher. A modified transposase enzymecontaining only a class 1 mutation can bind to the repeat sequences withsufficiently greater avidity than the wild-type Tn5 transposase enzymesuch that a Tn5 transposase enzyme induces about 5- to about 50-foldmore transposition than the wild-type transposase enzyme, when measuredin vivo. A modified transposase enzyme containing only a class 2mutation (e.g., a mutation that reduces the Tn5 transposase enzyme fromassuming an inactive form) is sufficiently less likely than thewild-type Tn5 transposase enzyme to assume the multimeric form that sucha Tn5 transposase enzyme also induces about 5-to about 50-fold moretransposition than the wild- type transposase enzyme, when measured invivo (See U.S. Pat. No. 5,965,443)

Other methods of using a modified transposase enzyme (e.g., modified Tn5transposase enzyme are further generally described in U.S. Pat. No.5,965,443. For example, a modified transposase enzyme could provideselective markers to target DNA, to provide portable regions of homologyto a target DNA, to facilitate insertion of specialized DNA sequencesinto target DNA, to provide primer binding sites or tags for DNAsequencing, or to facilitate production of genetic fusions for geneexpression. Studies and protein domain mapping, as well as, to bringtogether other desired combinations of DNA sequences (combinatorialgenetics) (U.S. Pat. No. 5,965,443).

Still other methods of inserting a transposable element (e.g.,transposon) at random or semi-random locations in chromosomal orextra-chromosomal nucleic acid are known. For example, methods includinga step of combining in a biological sample nucleic acid (e.g., genomicDNA) with a synaptic complex that comprises a Tn5 transposase enzymecomplexed with a sequence comprising a pair of nucleotide sequencesadapted for operably interacting with Tn5 transposase enzyme and atransposable element (e.g., transposon) under conditions that mediatetransposition events into the genomic DNA. In this method, a synapticcomplex can be formed in vitro under conditions that disfavor or preventsynaptic complexes from undergoing a transposition event. The frequencyof transposition (e.g., transposition events) can be increased by usingeither a hyperactive transposase enzyme (e.g., a mutant transposaseenzyme) or a transposable element (e.g., transposon) that containssequences well adapted for efficient transposition events in thepresence of a hyperactive transposase enzyme (e.g., hyperactive Tn5transposase enzyme), or both (U.S. Pat. No. 6,159,736, which isincorporated herein by reference).

Methods, compositions, and kits for treating nucleic acid, and inparticular, methods and compositions for fragmenting and tagging DNAusing transposon compositions are described in detail in U.S. Pat.Application Publication No. US 2010/0120098, U.S. Pat. ApplicationPublication No. US2011/0287435, and Satpathy, A.T., et. al., Massivelyparallel single-cell chromatin landscapes of human immune celldevelopment and intratumoral T-cell exhaustion, Nat Biotechnol., 37,925-936 (2019), the contents of which are herein incorporated byreference in their entireties.

Any transposase enzyme with tagmentation activity, e.g., any transposaseenzyme capable of fragmenting DNA and ligating oligonucleotides (e.g.,adapters, e.g. Nextera index adapters) to the ends of the fragmented(e.g., tagmented) DNA, can be used. In some embodiments, the transposaseis any transpose capable of conservative transposition. In someembodiments, the transposase is a cut and paste transposase. Other kindsof transposase are known in the art and are within the scope of thisdisclosure. For example, suitable transposase enzymes include, withoutlimitation, Mos-1, HyperMu™, Ts-Tn5, Ts-Tn5059, Hermes, Tn7, or anyfunctional variant or derivative of the previously listed transposaseenzymes.

In some embodiments, a hyperactive variant of the Tn5 transposase enzymeis capable of mediating the fragmentation of double-stranded DNA andligation of synthetic oligonucleotides (e.g., Nextera adapters) at both5′ ends of the DNA in a reaction that takes a short period of time(e.g., about 5 minutes). However, as wild-type end sequences have arelatively low activity, they are sometimes replaced in vitro byhyperactive mosaic end (ME) sequences. A complex of the Tn5 transposasewith 19-bp ME facilitates transposition, provided that the interveningDNA is long enough to bring two of these sequences close together toform an active Tn5 transposase enzyme homodimer.

In some embodiments, the Tn5 transposase enzyme, or functional variantor derivative thereof, comprises an amino acid sequence having at least80% sequence identity to SEQ. ID NO. 1. In some embodiments, the Tn5transposase enzyme, or functional variant or derivative thereof,comprises an amino acid sequence having a sequence identity of at leastabout 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% amino acid sequenceidentity to SEQ ID NO. 1.

In some embodiments, the transposase enzyme is a Mu transposase enzyme,or a functional variant or derivative thereof. In some embodiments, theMu transposase enzyme, or functional variant or derivative thereof,comprises an amino acid sequence having at least 80% sequence identityto SEQ. ID NO. 2. In some embodiments, the Mu transposase enzyme, orfunctional variant or derivative thereof, comprises an amino acidsequence having a sequence identity of at least about 90, 91, 92, 93,94, 95, 96, 97, 98, or 99% amino acid sequence identity to SEQ ID NO. 2.

The adaptors (e.g., Nextera adaptors) in the complex with thetransposase enzyme (e.g., that form part of the transposome, e.g., MEDSdescribed herein) can include partially double strandedoligonucleotides. In some embodiments, there is a first adapter and asecond adapter. In some embodiments, the first adapter can be complexedwith a first monomer. In some embodiments, the second adapter can becomplexed with a second monomer. In some embodiments, the first monomercomplexed with the first adapter and the second monomer complexed withthe second monomer can be assembled to form a dimer. In someembodiments, the double stranded portion of the adaptors contains MosaicEnd (ME) sequences. In some embodiments, the single stranded portion ofthe adaptors (e.g., Nextera index adapters) (5′ overhang) contains thefunctional domain or sequence to be incorporated in the fragmented(e.g., tagmented) DNA. In some embodiments, the adapters can be Nexteraadapters (e.g., index adapter) (for example, reagents including, NexteraDNA Library Prep Kit for ATAC-seq (no longer available), TDE-1 TagmentDNA Enzyme (Catalog No. 15027865), TD Tagment DNA Buffer (Catalog No.15027866), available from manufacturer, Illumina, Illumina.com). In someembodiments, the sequence incorporated into the fragmented (e.g.,tagmented) DNA is a sequence complementary to a capture domain of acapture probe. In some embodiments, the sequence complementary to thecapture domain of the capture probe is a first adapter. In suchembodiments, the functional domain is on the strand of the adaptor thatwill be ligated to the capture probe. In other words, the functionaldomain can be located upstream (e.g., 5′ to) the ME sequence, e.g., inthe 5′ overhang of the adapter.

The adaptors (e.g., Nextera index adapters, e.g., first and secondadapters) ligated to the fragmented (e.g., tagmented) DNA can be anysuitable sequence. For example, the sequence can be a viral sequence. Insome embodiments, the sequence can be a CRISPR sequence. In someembodiments, the adaptor (e.g., oligonucleotides) ligated to thefragmented DNA (e.g., tagmented DNA) can be a CRISPR guide sequence. Insome embodiments, the CRISPR guide sequence can target a sequence ofinterest (e.g., genomic locus of interest e.g., gene specific).

In some embodiments, the ME sequence is a Tn5 transposase recognitionsequence having at least 80% sequence identity to SEQ ID NO. 8. In someembodiments, the Tn5 transposase recognition sequence comprises asequence having a sequence identity of at least about 90, 91, 92, 93,94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO. 8, so long asthe Tn5 transposase enzyme, or variant or derivative, thereof canrecognize the Tn5 transposase sequence to induce a transposition event.

In some embodiments, the mosaic end (e.g., ME) sequence is a Mutransposase recognition sequence having at least 80% identity to any oneof SEQ ID NOs. 9-14. In some embodiments, the Mu transposase recognitionsequence comprises a sequence having a sequence identity of at leastabout 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity toany one of the sequence to which it is compared (e.g., any one of SEQ IDNOs. 9-14), so long as the Mu5 transposase enzyme, or variant orderivative thereof, can recognize the Mu5 transposase sequence to inducea transposition event.

In some embodiments, a composition comprising a transposase enzyme(e.g., any transposase enzyme described herein) complexed with adapters(e.g., first and second adapters complexed with first and secondmonomers, respectively) comprising transposon end sequences (e.g.,mosaic end sequences) is used in a method for spatially tagging nucleicacids in a biological sample. In some embodiments, a compositioncomprising a transposase enzyme further comprises a domain that binds toa capture probe as described herein (e.g., Nextera adapter, e.g., firstadapter) and a second adapter is used in a method for spatially taggingnucleic acids of a biological sample, such as any of the methodsdescribed herein.

In some embodiments, the transposase enzyme can be in the form of atransposome comprising adaptors (MEDS) in which the 5′ overhang can bephosphorylated. In some embodiments, the adaptors (e.g., Nexteraadaptors, e.g., first and second adapters) may be phosphorylated priorto their assembly with the transposase enzyme to form the transposome.In some embodiments, phosphorylation of adaptors can occur whencomplexed with a transposase enzyme (e.g., phosphorylation in situ inthe transposome).

As exemplified in the Examples provided herein, transposomes can includeadaptors (e.g., MEDS, e.g., adaptors including 5′ overhangs, e.g.,Nextera adaptors). In some embodiments, the 5′ overhang of the adaptoris not phosphorylated prior to its assembly in the transposome. In suchembodiments, the 5′ overhang can have accessible 5′ hydroxyl groupsoutside of the mosaic-end transposase sequence. In some embodiments,phosphorylation of the 5′ overhang of the assembled transposomecomplexes can be achieved by exposing these 5′ ends of transposomecomplexes to a polynucleotide kinase (e.g., T4-polynucleotide kinase(T4-PNK)) in the presence of ATP.

In some embodiments, fragmenting (e.g., tagmenting) genomic DNA of thebiological sample with a transposome (e.g., any of the transposomesdescribed herein) can comprise a further step of phosphorylating the 5′ends of the adaptors (e.g., the 5′ overhangs of the Nextera adaptors,e.g., MEDS) in the transposome complex.

In some embodiments, methods provided herein comprise a step ofproviding a transposome that has been treated to phosphorylate the 5′ends of the adaptors (e.g., the 5′ overhangs of the Nextera adaptors(e.g., first and second adapters), e.g., MEDS) in the transposomecomplex, thus fragmenting the biological sample with a transposome thathas been treated to phosphorylate the 5′ ends of the adaptors in thetransposome complex.

Any suitable enzyme and/or conditions can be used to phosphorylate the5′ ends of the adaptors (e.g., the 5′ overhangs of the adaptors, e.g.,MEDS) in the transposome complex, e.g., T4-PNK or T7-PNK. In someembodiments, the phosphorylation reaction can be carried out bycontacting the transposome with a polynucleotide kinase (e.g., T4-PNK orT7-PNK) in a buffered solution (e.g., Tris-HCl, pH about 7.0 to about8.0, e.g., about 7.6) at about 20 to about 40° C., e.g., about 25 toabout 37° C., for about 1 to about 60 minutes, e.g., about 5 to about50, about 10 to about 40, about 20 to about 30 minutes. In someembodiments, gap filling and ligating breaks can be performed on thefragmented (e.g., tagmented) DNA.

In some embodiments, spatially tagging the genomic DNA can be performedby insertion of the transposon sequence into the genomic DNA withadapters described herein. In some embodiments, the transposon sequenceis not excised from the genomic DNA. An amplification step can beperformed with primers to the adapters (e.g., inserted adapters into thegenomic DNA). The amplified products can contain accessible genomic DNAwhich can be spatially tagged by methods described herein.

In some embodiments, spatially tagging the genomic DNA can be performedby transposome complexes immobilized on the surface of the substrate. Insome embodiments, spatially tagging the genomic DNA can be performed bytransposome complexes immobilized on a feature (e.g., a bead). In someembodiments, the transposome complexes are assembled prior to adding thebiological sample to the substrate or features. In some embodiments, thetransposome complexes are assembled after adding the biological sampleto the substrate or features on a substrate. For example, a spatiallybarcoded substrate (e.g., array) can include a plurality of captureprobes that include a Mosaic End sequence (e.g., a transposaserecognition sequence). The Mosaic End sequence can be at the 3′ end ofthe capture probe (e.g., the capture probe is immobilized by its 5′ endand the Mosaic End sequence is at the 3′ most end of the capture probe).The Mosaic End sequence can be a Mosaic End sequence for any of thetransposase enzymes described herein. The Mosaic End sequence (e.g., atransposase recognition sequence) can be hybridized to a reversecomplement sequence (e.g., oligonucleotide). For example, the reversecomplement sequence (e.g., reverse complement to the Mosaic Endsequence) can hybridize to the Mosaic End sequence thereby generating aportion of double stranded DNA on the capture probe. The reversecomplement to the Mosaic End sequence (e.g., oligonucleotide) can beprovided to the spatially barcoded array prior to the biological samplebeing provided to the substrate. In some embodiments, the reversecomplement to the Mosaic End sequence can be provided after thebiological sample has been provided to the substrate. Transposaseenzymes can be provided to the substrate and assemble at the doublestranded portion of the capture probe (e.g., reverse complementoligonucleotide and the Mosaic End sequence hybridized to each other)thereby generating a transposome complex. For example, a transposomehomodimer can be formed at the double stranded portion of the captureprobe. A biological sample can be provided to the substrate such thatthe position of the capture probe on the substrate can be correlatedwith a position (e.g., location) in the biological sample. Thetransposome complexes can fragment (e.g., tagment) and spatially tag thegenomic DNA.

In some embodiments, spatially tagging genomic DNA can be performed byhybridizing a single stranded capture probe to the fragmented (e.g.,tagmented) DNA. In some embodiments the single stranded capture probecan be a degenerate sequence. In some embodiments, the single strandedcapture can be a random sequence. The single stranded capture probe canhave a functional domain, a spatial barcode, a unique molecularidentifier, a cleavage domain, or combinations thereof. The singlestranded capture probe (e.g., random sequence, degenerate sequence) cannon-specifically hybridize tagmented genomic DNA, thereby spatiallycapturing the fragmented (e.g., tagmented) DNA. Methods for extensionreactions are known in the art and any suitable extension reactionmethod described herein can be performed.

Splint Oligonucleotides

As used herein, the term “splint oligonucleotide” refers to anoligonucleotide that, when hybridized to other polynucleotides, acts asa “splint” (e.g., splint helper probe) to position the polynucleotidesnext to one another so that they can be ligated together. In someembodiments, the splint oligonucleotide is DNA or RNA. The splintoligonucleotide can include a nucleotide sequence that is partiallycomplementary to nucleotide sequences from two or more differentoligonucleotides. In some embodiments, the splint oligonucleotideassists in ligating a “donor” oligonucleotide and an “acceptor”oligonucleotide. In some embodiments, an RNA ligase, a DNA ligase, orother ligase can be used to ligate two nucleotide sequences together.

In some embodiments, the splint oligonucleotide can be between about 10and about 50 nucleotides in length, e.g., between about 10 and about 45,about 10 and about 40, about 10 and about 35, about 10 and about 30,about 10 and about 25, or about 10 and about 20 nucleotides in length.In some embodiments, the splint oligonucleotide can be between about 15and about 50, about 15 and about 45, about 15 and about 40, about 15 andabout 35, about 15 and about 30, about 15 and about 30, or about 15 andabout 25 nucleotides in length. In some embodiments, the fragmented DNAcan include a sequence that is added (e.g., ligated) duringfragmentation of the DNA. For example, during a transposition event(e.g., a Tn5 transposition event) an additional sequence can be attached(e.g., covalently attached, e.g., via a ligation event) to thefragmented DNA (e.g., fragmented genomic DNA, e.g., tagmented genomicDNA). In some embodiments, the splint oligonucleotide can have asequence that is complementary (e.g., a capture domain) to thefragmented DNA (e.g., fragmented genomic DNA, e.g., fragmented genomicDNA that includes a sequence that is added during fragmentation of theDNA, e.g. a first adapter attached during fragmentation of the DNA) anda sequence that is complementary to the surface probe (e.g., a portionof a capture probe). In some embodiments, the splint oligonucleotide canbe viewed as part of the capture probe. For example, the capture probecan be partially double stranded where a portion of the capture probecan function as a splint oligonucleotide that binds a portion of thecapture probe (e.g., dsDNA portion) and can have a single strand portionthat can bind (e.g., capture domain) the fragmented DNA (e.g.,fragmented genomic DNA e.g., tagmented, e.g., an adapter attached duringfragmentation of the DNA, e.g., a Nextera adapter). The first adaptersequence (e.g., the sequence attached to the fragmented DNAcomplementary to the capture domain, e.g., Nextera adapter) can be anysuitable sequence. In some embodiments, the adapter sequence can bebetween about 15 and 25 nucleotides long. In some embodiments, theadapter sequence can be about 16, about 17, about 18, about 19, about20, about 21, about 22, about 23, or about 24 nucleotides long. In someembodiments, the first adapter sequence (e.g., Nextera adapter) (e.g.,the first adapter) includes the sequence, GTCTCGTGGGCTCGG (SEQ ID NO:16). In some embodiments, the additional sequence attached to thefragmented (e.g., tagmented) DNA includes a sequence having at least 80%sequence identity to SEQ ID NO. 16. In some embodiments, the additionalsequence attached (e.g., Nextera adapter) to the fragmented DNA includesa sequence having at least about 90, 91, 92, 93, 94, 95, 96, 97, 98, or99% sequence identity to SEQ ID NO. 16. In some embodiments, a secondadapter sequence (e.g., Nextera adapter) can be attached to thefragmented DNA (e.g., tagmented DNA) that includes a sequence,TCGTCGGCAGCGTC (SEQ ID NO. 20). In some embodiments, the second adaptersequence attached (e.g., Nextera adapter) to the fragmented DNA (e.g.,tagmented DNA) includes a sequence having at least 80% sequence identityto SEQ ID NO. 20. In some embodiments, the second adapter sequence(e.g., Nextera adapter) attached to the fragmented (e.g., tagmented) DNAincludes a sequence having at least about 90, 91, 92, 93, 94, 95, 96,97, 98, or 99% sequence identity to SEQ ID NO. 20. In some embodiments,a splint oligonucleotide can include a sequence that is complementary(e.g., capture domain) to the first adapter attached to the fragmentedDNA (e.g., tagmented DNA). In some embodiments, the capture domain(e.g., complementary to the first adapter (e.g., Nextera adapter)) ofthe splint oligonucleotide (e.g., splint oligonucleotide of the captureprobe) can include the sequence CCGAGCCCACGAGAC (See FIG. 40 ; SEQ IDNO. 17). In some embodiments, the capture domain includes a sequencehaving at least 80% identity to SEQ ID NO. 17. In some embodiments, thecapture domain (e.g., sequence that is complementary to the firstadapter e.g., Nextera adapter) includes a sequence having at least about90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ IDNO. 17. In some embodiments, the splint oligonucleotide includes asequence that is not perfectly complementary to the first adapter (e.g.,Nextera adapter) attached to the fragmented DNA (e.g., tagmented DNA),but is still capable of hybridizing the first adapter sequence (e.g.,sequence complementary to the capture domain) ligated on to thefragmented DNA (e.g., Nextera adapter).

Any of a variety of capture probes having hybridization domains thathybridize to a splint oligonucleotide can be used in accordance withmaterials and methods described herein. As described herein, ahybridization domain is a domain on a surface probe capable ofhybridizing the splint oligonucleotide to form a partially doublestranded capture probe. For example, a single stranded surface probe canhave a sequence complementary (e.g., hybridization domain) to a portionof the splint oligonucleotide, such that a partially double strandedcapture probe is formed with a single stranded capture domain (e.g.,capture domain on the splint oligonucleotide). In some embodiments, thesurface probe (e.g., of the capture probe) can include a hybridizationdomain that includes the sequence TGCACGCGGTGTACAGACGT (SEQ ID NO. 18).In some embodiments, the surface probe (e.g., of the capture probe) caninclude a hybridization domain including a sequence having at least 80%identity to SEQ ID NO. 18. In some embodiments, the capture domainincludes a sequence having at least about 90, 91, 92, 93, 94, 95, 96,97, 98, or 99% sequence identity to SEQ ID NO. 18. In some embodiments,a splint oligonucleotide includes a sequence that is complementary(e.g., at least partially complementary) to the hybridization domain ofthe surface probe. In some embodiments, the sequence of the splintoligonucleotide (e.g., of the capture probe) that is complementary tothe hybridization domain of the surface probe (SEQ ID NO. 18) includesthe sequence ACGTCTGTACACCGCGTGCA (SEQ ID NO. 19). In some embodiments,the sequence of the splint oligonucleotide that is complementary to thecapture domain of the capture includes a sequence having at least 80%sequence identity to SEQ ID NO. 19. In some embodiments, the sequence ofthe splint oligonucleotide that is complementary (e.g., at leastpartially complementary) to the hybridization domain of the surfaceprobe includes a sequence having at least about 90, 91, 92, 93, 94, 95,96, 97, 98, or 99% sequence identity to SEQ ID NO. 19. In someembodiments, the splint oligonucleotide includes a sequence that is notperfectly complementary to the hybridization domain of the surfaceprobe, but is still capable of hybridizing the hybridization domain ofthe surface probe. In some embodiments, the splint oligonucleotide canhybridize to both the first adapter (e.g., additional sequence attachedto the fragmented DNA e.g., tagmented DNA) via its capture domain andthe hybridization domain of the surface probe via its sequencecomplementary to the hybridization domain. In such embodiments, wherethe splint oligonucleotide can hybridize to both the first adapter(e.g., Nextera adapter, additional sequence attached to the fragmentedDNA e.g., tagmented DNA), and the hybridization domain of the surfaceprobe, the splint oligonucleotide can be viewed as part of the captureprobe. In some embodiments, a primer can have a sequence capable ofhybridizing the surface probe (e.g., surface probe of the capture probe)sequence. For example, the primer can have a sequence that includes thesequence ACACGACGCTCTTCCGATCT (SEQ ID NO. 21). In some embodiments, thesequence that is capable of hybridizing a portion of the surface probeof the capture probe (e.g., A-short forward, See FIG. 40 ) includes asequence having at least 80% sequence identity to SEQ ID NO. 21. In someembodiments, the sequence that is complementary (e.g., at leastpartially complementary) to a portion of the capture probe (e.g.,A-short forward) includes a sequence having at least about 90, 91, 92,93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO. 21.

In some embodiments, the splint oligonucleotide can have a capturedomain that is homopolymeric. For example, the capture domain can be apoly(T) capture domain.

In some embodiments, a splint oligonucleotide can facilitate ligation ofthe fragmented DNA (e.g. tagmented DNA) and the surface probe. Anyvariety of suitable ligases known in the art or described herein can beused. In some embodiments, the ligase is T4 DNA ligase. In someembodiments, the ligation reaction can last for about 1 to about 5hours. In some embodiments, the ligation reaction can last for about 2,about 3, or about 4 hours. In some embodiments, after ligation, stranddisplacement polymerization can be performed. In some embodiments, a DNApolymerase can be used to perform the strand displacementpolymerization. In some embodiments, the DNA polymerase is DNApolymerase I.

Multiplex Analysis

The present disclosure describes methods for permeabilizing biologicalsamples under conditions sufficient to allow fragmentation (e.g.,tagmentation) of genomic DNA. The fragmented (e.g., tagmented) DNA canbe captured via a capture probe (e.g., surface probe and a splintoligonucleotide), however, at times it can be useful to simultaneouslycapture fragmented (e.g., tagmented DNA) and other nuclei acids (e.g.,mRNA). For example, expression profiles of transcripts can be correlated(or not) with open chromatin. Put another way, the presence oftranscripts can correlate with open chromatin (e.g., accessiblechromatin) corresponding to the genes (e.g., genomic DNA) from which thetranscripts were transcribed.

The present disclosure describes methods regarding the simultaneouscapture of fragmented DNA (e.g., tagmented DNA) and mRNA on spatiallybarcoded arrays. For example, a spatially barcoded array can have aplurality of capture probes immobilized on a substrate surface.Alternatively, a spatially barcoded array can have a plurality ofcapture probes immobilized on a feature. In some embodiments, thefeature with a plurality of capture probes can be on a substrate. Thecapture probes can have unique spatial barcodes corresponding to aposition (e.g., location) on the substrate. In some embodiments, thecapture probes can further have a unique molecular identifier,functional domain, and a cleavage domain, or combinations thereof. Insome embodiments, the capture probe can have a capture domain. In someembodiments, the capture probe can be a homopolymeric sequence. Forexample, in a nonlimiting way, the homopolymeric sequence can be apoly(T) sequence. In some embodiments, nucleic acid (e.g., mRNA) can becaptured by the capture domain by binding (e.g., hybridizing) of poly(A)tails of mRNA transcripts. In some embodiments, fragmented DNA (e.g.,tagmented DNA) can be captured by the capture domain of the captureprobe by binding (e.g., hybridizing) a poly(A) tailed fragmented DNA(e.g., tagmented DNA). For example, after fragmenting the genomic DNA,gap filing (e.g., no strand displacement) polymerases and ligases canrepair gaps and ligate breaks in the fragmented (e.g., tagmented DNA).In some embodiments, a sequence complementary to the capture domain canbe introduced to the fragmented DNA. For example, a poly(A) tail can beadded to the fragmented (e.g., tagmented) DNA, such that the capturedomain (e.g., poly(T) sequence) of the capture probe can bind (e.g.,hybridize) to the poly(A) tailed fragmented (e.g., tagmented DNA) (See,e.g., WO 2012/140224, which is incorporated herein by reference). Insome embodiments, a poly(A) tail could be added to the fragmented DNA(e.g., tagmented) by a terminal transferase enzyme. In some embodiments,the terminal transferase enzyme could be terminal deoxynucleotidyltransferase (TDT), or a mutant variant thereof. TDT is an independentpolymerase (e.g., it does not require a template molecule) that cancatalyze the addition of deoxynucleotides to the 3′ hydroxyl terminus ofDNA molecules. Other template independent polymerases are known in theart. For example, Polymerase Π, or a mutant variant thereof, may be usedas a terminal transferase enzyme (See, e.g., Kent, T., Polymerase Π is arobust terminal transferase that oscillates between three differentmechanisms during end-joining, eLIFE, 5: e13740 doi:10.7554/eLife.13740, (2016)). Other methods of introducing a poly(A)tail are known in the art. In some embodiments, a poly(A) tail can beintroduced to the fragmented DNA (e.g., tagmented DNA) by a non-proofreading polymerase. In some embodiments, a poly(A) tail can beintroduced to the fragmented DNA by a polynucleotide kinase.

In some embodiments, the TDT enzyme will generate fragments (e.g.,tagments) with a 3′ poly(A) tail, thereby mimicking the poly(A) tail ofan mRNA. In some embodiments, the capture domain (e.g., poly(T)sequence) of the capture probe would interact with the poly(A) tail ofthe mRNA and the generated (e.g., synthesized) poly(A) tail added to thefragmented (e.g., tagmented) DNA, thereby simultaneously capturing thefragmented DNA (e.g., tagmented DNA) and the mRNA transcript. Thegenerated (e.g., synthesized) poly(A) tail on the fragmented DNA (e.g.,tagmented DNA) could be between about 10 nucleotides to about 30nucleotides long. The generated (e.g., synthesized) poly(A) tail on thefragmented DNA (e.g., tagmented DNA) could be about 11, about 12, about13, about 14, about 15, about 16, about 17, about 18, about 19, about20, about 21, about 22, about 23, about 24, about 25, about 26, about27, about 28, or about 29 nucleotides long.

Additionally and alternatively, instead of a sequential (e.g. two-step)reaction (e.g., gap filling and ligating, followed by a terminaltransferase) the fragmented (e.g., tagmented) DNA can be contacted witha polymerase. For example, the polymerase may be a DNA polymerase thatmay perform an extension reaction on the fragmented (e.g., tagmentedDNA. Any variety of DNA polymerases known in the art or described hereincan be used. The extended products can be captured and processed (e.g.,amplified and sequenced) by any method described herein.

Post-hybridization steps are identical as described in Ståhl P.L., etal., Visualization and analysis of gene expression in tissue sections byspatial transcriptomics Science, vol. 353, 6294, pp. 78-82 (2016), whichin incorporated herein by reference).

qPCR and Analysis

Also provided herein are methods and materials for quantifying captureefficiency. In some embodiments, quantification of capture efficiencyincludes quantification of captured fragments (e.g., genomic DNAfragments, e.g., tagmented DNA fragments) from any of the spatialanalysis methods described herein. In some embodiments, quantificationincludes PCR, qPCR, electrophoresis, capillary electrophoresis,fluorescence spectroscopy and/or UV spectrophotometry. In someembodiments, qPCR includes intercalating fluorescent dyes (e.g., SYBRgreen) and/or fluorescent labeled-probes (e.g., without limitation,Taqman probes or PrimeTime probes). In some embodiments, a NGS libraryquantification kit is used for quantification. For example,quantification can be performed using a KAPA library quantification kit(KAPA Biosystems), qPCR NGS Library Quantification Kit (Agilent),GeneRead Library Quant System (Qiagen), and/or PerfeCTa NGSQuantification Kit (Quantabio). In some embodiments that use qPCR forquantification, qPCR can include, without limitation, digital PCR,droplet digital (ddPCR), and ddPCR-Tail. In some embodiments that useelectrophoresis for quantification, electrophoresis can include, withoutlimitation, automated electrophoresis (e.g., TapeStation System,Agilent, and/or Bioanalzyer, Agilent) and capillary electrophoresis(e.g., Fragment Analyzer, Applied Biosystems). In some embodiments thatuse spectroscopy for quantification, the spectroscopy can include,without limitation, fluorescence spectroscopy (e.g., Qubit, ThermoFisher). In some embodiments, NGS can be used to quantify captureefficiency.

In some embodiments, quantitative PCR (qPCR) is performed on thecaptured tagments. In some embodiments, the fragmented (e.g., tagmented)DNA is amplified, by any method described herein, before capture. Forexample, after capture of the fragmented DNA (e.g., tagmented DNA),ligation and strand displacement hybridization qPCR can be performed. Insome embodiments, a DNA polymerase can be used to perform the stranddisplacement polymerization. Any suitable strand displacement polymeraseknown in the art can be used. In some embodiments, the DNA polymerase isDNA polymerase I. As exemplified in the Examples, DNA polymerase I canbe incubated for strand displacement of the fragmented DNA (e.g.,tagmented DNA) with reagents (e.g., BSA, dNTPs, buffer). In someembodiments, DNA polymerase I can be incubated with reagents on thesubstrate (e.g., on a feature e.g., a well) for about 30 minutes toabout 2 hours. In some embodiments, DNA polymerase I can be incubatedwith reagents on the substrate for about 40 minutes, about 50 minutes,about 60 minutes, about 70 minutes, about 80 minutes, about 90 minutes,about 100 minutes, or about 110 minutes. In some embodiments, DNApolymerase I can be incubated with reagents on the substrate (e.g., on afeature e.g., a well) at about 35° C. to about 40° C. In someembodiments, DNA polymerase I can be incubated with reagents on thesubstrate at about 36° C., about 37° C., about 38° C., or about °C, orabout 39° C. In some embodiments, DNA polymerase I can be incubated withreagents on the substrate for about 1 hour at about 37° C.

After strand displacement hybridization is complete a qPCR reaction canbe performed. As exemplified in the Examples below, the capture probesligated to the fragmented DNA (e.g., tagmented DNA), can be releasedfrom the surface of the substrate (e.g., feature). In some embodiments,a solution (e.g., release mix) can be incubated with the substrate torelease the capture probes from the surface of the substrate. Therelease mix can contain reagents (e.g., BSA, enzymes, buffer). Methodsof releasing capture probes from the substrate (e.g., a feature) aredescribed herein. In some embodiments, an enzyme can cleave the captureprobe. In some embodiments, the enzyme can be USER (uracil-specificexcision reagent) enzyme. In some embodiments, the USER enzyme can beincubated with reagents on the substrate (e.g., a feature e.g., a well)for about 30 minutes to about 2 hours. In some embodiments, the USERenzyme can be incubated with reagents on the substrate for about 40minutes, about 50 minutes, about 60 minutes, about 70 minutes, about 80minutes, about 90 minutes, about 100 minutes, or about 110 minutes. Insome embodiments, the USER enzyme with reagents on the substrate (e.g.,a feature e.g., a well) at about 35° C. to about 40° C. In someembodiments, the USER enzyme can be incubated with reagents on thesubstrate at about 36° C., about 37° C., about 38° C., or about 39° C.In some embodiments, the USER enzyme can be incubated with reagents onthe substrate for about 1 hour at about 37° C.

After incubation with the USER enzyme, the samples (e.g., releasedcapture probes ligated to fragmented DNA (e.g., tagmented DNA) inrelease mix, or a portion thereof) can be collected. In someembodiments, the sample volume can be reduced. Methods of reducingsample volume are known in the art and any suitable method can be used.In some embodiments, sample volume reduction can be performed with aSpeed Vacuum (e.g., a SpeedVac). In some embodiments, the sample volumereduction can be about 50, about 55, about 60, about 65, about 70, about75, about 80, about 85, or about 90% sample volume reduction. In someembodiments, the sample volume reduction can be about between 80% and90% sample volume reduction. In some embodiments, the sample volumereduction can be about 81, about 82, about 83, about 84, about 85, about86, about 87, about 88, or about 89% sample volume reduction. In someembodiments, the sample volume reduction can be about 85% (e.g., about10 µL after sample volume reduction).

In some embodiments, a qPCR reaction can be performed with the reducedsample volume. As described herein, any suitable method of qPCR can beperformed. As exemplified in the Examples, a 1xKAPA HiFI HotStart Ready,1x EVA green, and primers can be used. Amplification can be performedaccording to known methods in the art. For example, amplification can beperformed accordingly: 72° C. for 10 minutes, 98° C. for 3 minutes,followed by cycling at 98° C. for 20 seconds, 60° C. for 30 seconds and72° C. for 30 seconds.

In some embodiments, one or more primer pairs can be used during theqPCR reaction. As described in the Examples herein, a primer pair cancover the ligated portion (e.g., ligation site where the capture probeand adapter sequence (e.g., attached sequence to the fragmented DNAe.g., tagmented DNA)). For example, a primer pair, (A-short forward andNextera reverse (FIG. 40 ); SEQ ID NOs. 21 and 20, respectively) coversthe ligated portion and the capture probe. An amplification product willonly be detected if ligation, and not just hybridization has occurred.In some embodiments, a different primer pair (e.g., Nextera forward andNextera reverse (FIG. 40 ); SEQ ID NOs. 16 and 20, respectively) cancover the fragmented DNA (e.g. tagmented DNA) only. In some embodiments,the primer pair that covers the fragmented DNA (e.g., tagmented DNA)only can be a control for ligation. In some embodiments, qPCR can beperformed with any of labeled nucleotides described herein.

In some embodiments, the samples can be purified. In some embodiments,the samples can be purified according to Lundin et al., IncreasedThroughput by Parallelization of Library Preparation for MassiveSequencing, PLOS ONE, 5(4), doi.org/10.1371/journal.pone.0010029 (2010),which is herein incorporated by reference.

In some embodiments, the average length of the captured fragmented DNA(e.g., tagmented DNA) can be determined. In some embodiments, abioanalyzer (e.g., a 2100 Bioanalyzer (Agilent)) can be used. Anysuitable bioanalyzer known in the art can be used. In some embodiments,qPCR and bioanalyzer analysis can be done on whole genomes (e.g.,purified fragmented DNA e.g., tagmented DNA). In some embodiments, theqPCR and bioanalyzer analysis can be done on an immobilized biologicalsample (e.g., a fixed biological sample). For example, the methodsdescribed herein (e.g., pre-permeabilization, permeabilization) can beperformed to capture fragmented DNA (e.g., tagmented DNA) and tooptimize qPCR and bioanalyzer analysis for different biological samples.

In some embodiments, after ligation, a surface based denaturation stepcan be performed. Put another way, after ligation of the fragmented DNA(e.g., tagmented DNA) to the capture probe, followed by stranddisplacement hybridization described herein (e.g., DNA Polymerase I), asurface based denaturation step can be performed in a parallelworkstream. In some embodiments, a basic solution can perform thesurface based denaturation. For example, the basic solution can denaturethe captured double stranded fragmented DNA (e.g., tagmented DNA), thusgenerating captured single stranded capture probes ligated to fragmentedDNA (e.g., tagmented DNA). In some embodiments, the basic solution canbe about 1 M NaOH. Other basic solutions can be used in the methodsdescribed herein. In some embodiments, the basic solution can be appliedfor about 1 minute to about 1 hour. In some embodiments, the basicsolution can be applied for about 10, about 20, about 30, about 40, orabout 50 minutes. In some embodiments, the basic solution can be appliedfor about 1 to about 20 minutes. In some embodiments, the basic solutionabout be applied for about 2, about 3, about 4, about 5, about 6, about7, about 8, about 9, about 10, about 11, about 12, about 13, about 14about 15, about 16, about 17, about 18, or about 19 minutes. In someembodiments, the basic solution can be applied at a temperature ofbetween about 30° C. to about 40° C. In some embodiments, the basicsolution can be applied at about 31° C., about 32° C., about 33° C.,about 34° C., about 35° C., about 36° C., about 37° C., about 38° C., orabout 39° C. In some embodiments, the basic solution can be applied forabout 10 minutes at about 37° C.

In some embodiments, the denaturation step can expose the fragmented DNA(e.g., tagmented DNA) to hybridization by a probe. In some embodiments,the probe can be an oligonucleotide probe. In some embodiments, theoligonucleotide probe can have a detectable label (e.g., any of thevariety of detectable labels described herein). In some embodiments, thedetectable label can be Cy5. In some embodiments, the oligonucleotideprobe can be Cy5 labeled. In some embodiments, the Cy5 labeledoligonucleotide probe can hybridize to a complementary sequence in thefragmented DNA (e.g., tagmented DNA). In some embodiments, the Cy5labeled oligonucleotide can hybridize to the sequence attached (e.g.,Nextera adapter, e.g., first adapter or second adapter) to thefragmented DNA (e.g., tagmented DNA). In some embodiments, the Cy5 labelcan be detected. For example, detecting the Cy5 label in theoligonucleotide probe can reveal the spatial location of the DNAtagments. In some embodiments, the biological sample can be stained(e.g., hematoxylin and eosin stain). Methods of staining a biologicalsample are known in the art and described herein. In some embodiments,the biological sample can be imaged.

Embodiments

Accordingly, in one embodiment the present invention provides a methodfor spatially tagging nucleic acids of a biological specimen comprising:

-   (a) providing a solid substrate on which multiple species of capture    probes are immobilized such that each species occupies a distinct    position on the solid substrate, wherein said capture probes are for    an extension or ligation reaction and wherein each species of said    capture probes comprise a nucleic acid molecule comprising:    -   (i) a positional domain that corresponds to the position of the        capture probe on the solid substrate, and    -   (iii) a capture domain;-   (b) contacting said solid substrate with a biological specimen;-   (c) permeabilizing the biological specimen under conditions    sufficient to make DNA in the biological specimen accessible to a    transposase enzyme;-   (d) fragmenting the DNA in said biological specimen with a    transposase enzyme;-   (e) hybridizing the fragmented DNA present in the biological    specimen from (d) to the capture domains of the capture probes; and-   (f) extending the capture probes:    -   (i) using the DNA hybridized to said capture probes as extension        or ligation templates to produce extended probes that comprise        the sequences of the positional domains and sequences        complementary to the DNA that hybridizes to the capture domains        of the capture probes; or    -   (ii) using the capture probes as ligation templates to produce        extended probes that comprise the sequence of the positional        domains or a complement thereof and sequences of the DNA that        hybridize to the capture domains of the capture probes, thereby        spatially tagging the DNA of the biological specimen.

As discussed in more detail below, the method of the invention maycomprise an additional step of analysing the extended probes. In thisrespect, it is evident that the combination of spatial tagging of thenucleic acids from a biological specimen and subsequent analysis of saidtagged nucleic acids facilitates the localised detection of a nucleicacid in a biological specimen, e.g. tissue sample. Thus, in oneembodiment, the method of the invention may be used for determiningand/or analysing all of the genome or the genome and transcriptome of abiological specimen. However, the method is not limited to this andencompasses determining and/or analysing all or part of the genome orall of part of the genome and transcriptome. Thus, the method mayinvolve determining and/or analysing a part or subset of the genome orgenome and transcriptome, e.g. a genome corresponding to a subset ofgenes, e.g. a set of particular genes, for example related to aparticular disease or condition, tissue type etc.

In other embodiments, the invention provides a method for spatiallytagging nucleic acids of a biological specimen comprising:

-   (a) providing a solid substrate comprising a plurality of capture    probes attached to the solid substrate, wherein a capture probe of    the plurality of capture probes comprises a capture domain and a    position domain, wherein the position domain corresponds to a    distinct position on the solid substrate;-   (b) contacting said solid substrate with a biological specimen;-   (c) permeabilizing the biological specimen under conditions    sufficient to make DNA in the biological specimen accessible to a    transposase enzyme;-   (d) fragmenting the DNA in said biological specimen with the    transposase enzyme;-   (e) contacting the fragmented DNA present in the biological specimen    from (d) to the capture domains of the capture probes; and-   (f) extending the capture probes,-   thereby spatially tagging the DNA of the biological specimen.

In some embodiments, step (e) of contacting the fragmented DNA comprises(i) using the DNA contacted with said capture probes as extension orligation templates to produce extended probes that comprise thesequences of the positional domains and sequences complementary to theDNA that hybridizes to the capture domains of the capture probes, (ii)using the capture probes as ligation templates to produce extendedprobes that comprise the sequence of the positional domains or acomplement thereof and sequences of the DNA that hybridizes to thecapture domains of the capture probes.

Viewed from another aspect, the method steps set out above can be seenas providing a method of obtaining a spatially defined genome or genomeand transcriptome, and in particular the spatially defined global genomeor genome and transcriptome of a biological specimen, e.g. tissuesample.

Alternatively viewed, the method of the invention may be seen as amethod for localised or spatial detection of nucleic acid, whether DNAor both DNA and RNA, in a biological specimen, e.g. tissue sample, orfor localised or spatial determination and/or analysis of nucleic acid(DNA or both DNA and RNA) in a tissue sample. In particular, the methodmay be used for the localised or spatial detection or determinationand/or analysis of genomic variation or genomic variation and geneexpression in a tissue sample. The localised/spatialdetection/determination/analysis means that the DNA or both DNA and RNAmay be localised to its native position or location within a cell ortissue in the tissue sample. Thus for example, the DNA or both DNA andRNA may be localised to a cell or group of cells, or type of cells inthe sample, or to particular regions of areas within a tissue sample.The native location or position of the DNA or DNA and RNA (or in otherwords, the location or position of the DNA or DNA and RNA in the tissuesample), e.g. a genomic locus or genomic locus and expressed gene, maybe determined.

Thus, in some embodiments, the invention provides a method for localiseddetection of nucleic acid in a biological specimen comprising:

-   (a) providing a solid substrate on which multiple species of capture    probes are immobilized such that each species occupies a distinct    position on the solid substrate, wherein said capture probes are for    an extension or ligation reaction and wherein each species of said    capture probes comprise a nucleic acid molecule comprising:    -   (i) a positional domain that corresponds to the position of the        capture probe on the solid substrate, and    -   (ii) a capture domain;-   (b) contacting said solid substrate with a biological specimen;-   (c) permeabilizing the biological specimen under conditions    sufficient to make DNA in the biological specimen accessible to a    transposase enzyme;-   (d) fragmenting the DNA in said biological specimen with the    transposase enzyme;-   (e) hybridizing the fragmented DNA present in the biological    specimen from (d) to the capture domains of the capture probes; and-   (f) extending the capture probes:    -   (i) using the DNA hybridized to said capture probes as extension        or ligation templates to produce extended probes that comprise        the sequences of the positional domains and sequences        complementary to the DNA that hybridizes to the capture domains        of the capture probes; or    -   (ii) using the capture probes as ligation templates to produce        extended probes that comprise sequences of the positional        domains or complements thereof and sequences of the DNA that        hybridizes to the capture domains of the capture probes,    -   thereby spatially tagging the DNA of the biological specimen;        and-   (g) analysing the extended probes of (f), i.e. analysing the    spatially tagged nucleic acids of the biological specimen.

The method may further comprise a step of releasing the extended probesof (f) from the surface of the solid substrate, i.e. extended probesthat comprise the sequences of the positional domains and sequencescomplementary to the nucleic acids that hybridize to the capture domainsof the capture probes or extended probes that comprise sequences ofpositional domains or complements thereof and sequences of the DNA thathybridizes to the capture domains of the capture probes. As discussed inmore detail below, the extended probes may be released from the surfaceof the substrate by any suitable means. In some embodiments, theextended probes may be released prior to the analysis step (step (g)),but this is not essential. For instance, the extended probes may bereleased from the surface of the substrate as part of the analysis step.

Any method of nucleic acid analysis may be used in the analysis step(step (g)). Typically this may involve sequencing, i.e. analysing thesequence of the extended probes, but it is not necessary to perform anactual sequence determination. For example sequence-specific methods ofanalysis may be used. For example a sequence-specific amplificationreaction may be performed, for example using primers which are specificfor the positional domain and/or for a specific target sequence, e.g. aparticular target DNA to be detected (i.e. corresponding to a particularcDNA/RNA and/or gene, intergenic or intragenic region etc.). Anexemplary analysis method is a sequence-specific PCR reaction.

The sequence analysis information obtained in step (g) may be used toobtain spatial information as to the DNA and/or RNA in the biologicalspecimen, e.g. tissue sample. In other words the sequence analysisinformation may provide information as to the location of the DNA and/orRNA in the biological specimen, e.g. tissue sample. This spatialinformation may be derived from the nature of the sequence analysisinformation determined, for example it may reveal the presence of aparticular DNA and/or RNA which may itself be spatially informative inthe context of the biological specimen, e.g. tissue sample, used, and/orthe spatial information (e.g. spatial localisation) may be derived fromthe position of the biological specimen, e.g. tissue sample, on thesolid substrate, e.g. array, coupled with the sequencing information.Thus, the method may involve simply correlating the sequence analysisinformation to a position in the biological specimen, e.g. tissuesample, e.g. by virtue of the positional tag and its correlation to aposition in the biological specimen, e.g. tissue sample. However, insome embodiments, spatial information may conveniently be obtained bycorrelating the sequence analysis data to an image of the biologicalspecimen, e.g. tissue sample. Accordingly, in a preferred embodiment themethod also includes a step of:

(h) correlating said sequence analysis information with an image of saidbiological specimen, wherein the biological specimen is imaged afterstep (b). In some embodiments, the biological specimen is imaged beforestep (c) or (d).

It will be seen therefore that the array of the present invention may beused to capture DNA (e.g. genomic DNA) or both DNA and RNA (e.g. mRNA)of a biological specimen, e.g. tissue sample, that is contacted withsaid solid substrate, e.g. array. The methods of the invention may thusbe considered as methods of quantifying the spatial variation of one ormore genes in a tissue sample (e.g. copy number variation). Expressedanother way, the methods of the present invention may be used to detectthe spatial variation of one or more genes in a biological specimen,e.g. tissue sample. In yet another way, the methods of the presentinvention may be used to determine simultaneously the variation of oneor more genes at one or more positions within a biological specimen,e.g. tissue sample. Still further, the methods may be seen as methodsfor partial or global genome or genome and transcriptome analysis of abiological specimen, e.g. tissue sample, with two-dimensional spatialresolution.

It will be evident that when the method of the invention is used toanalyse DNA or both DNA and RNA in a tissue section of a biologicalspecimen to yield a two-dimensional genome or genome and transcriptome,data from analyses of other tissue sections from the same biologicalspecimen (tissue sample), particularly adjacent tissue sections, may becompiled to provide a three-dimensional genome or genome andtranscriptome of the biological specimen.

Thus, at its broadest, the present invention may be viewed as the use oftagmentation in an immobilized biological specimen (e.g. a tissuesection on a solid substrate) to facilitate the spatial tagging of DNAin the biological specimen, preferably using a method as defined herein.

In another aspect, the invention provides a kit for use in the methodsdescribed herein. The kit may comprise any two or more of:

-   (i) a solid substrate (e.g. array) on which multiple species of    capture probes are immobilized as defined above;-   (ii) means for permeabilizing a biological specimen to make it    accessible to a transposase enzyme, particularly enzymatic or    chemical means as defined herein;-   (iii) means for tagmenting DNA in a biological specimen,    particularly a transposome as defined herein;-   (iv) means for extending the capture probes, such as a reverse    transcriptase, DNA polymerase, DNA ligase or a mixture thereof as    defined above; and-   (v) means for releasing the extended probes from the solid    substrate, particularly a cleavage enzyme or mixture thereof as    defined above.

In some embodiments, the kit may additionally or alternatively comprisecomponents for use with means defined above, e.g. buffers and substrates(e.g. dNTPs) suitable for the enzymes defined above. In someembodiments, the kit may comprise means for generating second strand DNAmolecules (e.g. helper probes, primers, adaptors etc) and/or foramplifying the extended probes (e.g. DNA polymerases, primers,substrates, buffers etc.).

In some embodiments, the kit may comprise components for producing thesolid substrate. For instance, the solid substrate may be provided withsurface probes and the kit may comprise reagents for producing thecapture probes of the invention, e.g. capture domain oligonucleotides.In some embodiments, the kit comprises a solid substrate and means forgenerating capture probes using bridge amplification as described above.In some embodiments, the kit may comprise means for generating a beadarray for use in the methods of the invention as described above, e.g. asolid substrate on which beads may be immobilized and beads on whichcapture probes of the invention are immobilized. In some embodiments,the kit may comprise means for decoding an array, e.g. decoder probes asdescribed above.

In some embodiments, the kit may comprise means for fixing and/orstaining the biological specimen.

In some embodiments, the kit may comprise means for purifying extendedprobes and/or their amplicons that have been released from the surfaceof the substrate.

“Tagmentation” refers to a process of transposase-mediated fragmentationand tagging of DNA. Tagmentation typically involves the modification ofDNA by a transposome complex and results in the formation of “tagments”,or tagged DNA fragments.

A “transposome” or “transposome complex” is a complex of a transposaseenzyme and DNA which comprises a transposon end sequence (also known as“transposase recognition sequence” or “mosaic end” (ME)).

The DNA that forms a complex with a transposase enzyme (i.e. the DNA ofa transposome) contains a partially double stranded (e.g. DNA)oligonucleotide, wherein each strand contains an ME specific for thetransposase, which forms the double stranded part of theoligonucleotide. The single-stranded portion of the oligonucleotide isat the 5′ end of the oligonucleotide (i.e. forms a 5′ overhang) and maycomprise a functional sequence (e.g. a capture probe binding site).Thus, the partially double stranded oligonucleotide in the transposomemay be viewed as an adaptor that can be ligated to the fragmented DNA.Thus, alternatively viewed the transposome comprises a transposaseenzyme complexed with an adaptor comprising transposon end sequences (ormosaic ends) and tagmentation results in the simultaneous fragmentationof DNA and ligation of the adapters to the 5′ ends of both strands ofDNA duplex fragments.

Thus, alternatively viewed step (d) may be viewed as tagmenting the DNAof the biological specimen comprising contacting the biological specimenwith a transposome, i.e. under conditions sufficient to result intagmentation of the DNA.

It will be evident that tagmentation can be used to provide fragmentedDNA with a binding domain capable of binding (hybridizing) to thecapture domain of the capture probes of the invention. Moreover, thebinding domain may be provided directly or indirectly.

Thus, in some embodiments, step (d) may be viewed as fragmenting the DNAof the biological specimen and providing the DNA fragments with abinding domain capable of binding (hybridizing) to the capture domain ofthe capture probes of the invention.

For example, in some embodiments, the adaptors of the transposomecomprise a functional domain or sequence that may be configured tocouple to all or a portion of a capture domain. The functional domain orsequence which may be a binding domain capable of binding (hybridizing)to the capture domain of the capture probes of the invention (e.g. ahomopolymeric sequence, e.g. poly-A sequence, as defined below). Inother words, the single-stranded portion of the adaptor (5′ overhang)comprises a binding domain capable of binding to the capture domain ofthe capture probes of the invention. Accordingly, tagmentation resultsfragmentation of DNA of the biological specimen and ligation of thebinding domain capable of binding to the capture domain of the captureprobes of the invention to the DNA of the biological specimen, i.e.providing the DNA of the biological specimen with a binding domaindirectly.

In one embodiment, the functional domain or sequence is configured tocouple to or attach to a portion of the capture domain through clickchemistry. As used herein, the term “click chemistry,” generally refersto reactions that are modular, wide in scope, give high yields, generateonly inoffensive byproducts, such as those that can be removed bynonchromatographic methods, and are stereospecific (but not necessarilyenantioselective). See, e.g., Angew. Chem. Int. Ed., 2001,40(11):2004-2021, which is entirely incorporated herein by reference forall purposes. In some cases, click chemistry can describe pairs offunctional groups that can selectively react with each other in mild,aqueous conditions.

An example of click chemistry reaction can be the Huisgen 1,3-dipolarcycloaddition of an azide and an alkyne, i.e., Copper-catalysed reactionof an azide with an alkyne to form a 5-membered heteroatom ring called1,2,3-triazole. The reaction can also be known as a Cu(I)-CatalyzedAzide-Alkyne Cycloaddition (CuAAC), a Cu(I) click chemistry or a Cu+click chemistry. Catalyst for the click chemistry can be Cu(I) salts, orCu(I) salts made in situ by reducing Cu(II) reagent to Cu(I) reagentwith a reducing reagent (Pharm Res. 2008, 25(10): 2216-2230). KnownCu(II) reagents for the click chemistry can include, but are not limitedto, Cu(II)-(TBTA) complex and Cu(II) (THPTA) complex. TBTA, which istris-[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine, also known astris-(benzyltriazolylmethyl)amine, can be a stabilizing ligand for Cu(I)salts. THPTA, which is tris-(hydroxypropyltriazolylmethyl)amine, can beanother example of stabilizing agent for Cu(I). Other conditions canalso be accomplished to construct the 1,2,3-triazole ring from an azideand an alkyne using copper-free click chemistry, such as by theStrain-promoted Azide-Alkyne Click chemistry reaction (SPAAC, see, e.g.,Chem. Commun., 2011, 47:6257-6259 and Nature, 2015, 519(7544):486-90),each of which is entirely incorporated herein by reference for allpurposes.

Thus, in some embodiments, step (d) may be viewed as contacting thebiological specimen with a transposase complexed with an adaptorcomprising transposon end (e.g. mosaic end) sequences (i.e. atransposome) and a nucleotide sequence that is complementary to thecapture domain of the capture probes and wherein the transposase ligatesthe adaptor to the fragmented DNA, i.e. the 5′ ends of the fragmentedDNA.

In other embodiments, step (d) may be viewed as contacting thebiological specimen with a transposase complexed with an adaptorcomprising transposon end (e.g. mosaic end) sequences (i.e. atransposome) and a click chemistry moiety(ies) that is compatible withanother click chemistry moiety(ies) on the capture domain of the captureprobes and wherein the transposase ligates the adaptor to the fragmentedDNA, i.e., the 5′ ends of the fragmented DNA.

In some embodiments, the adaptor of the transposome comprises (i) adomain capable of (i.e. suitable for) facilitating the introduction of abinding domain capable of binding (hybridizing) to the capture domain ofthe capture probes of the invention or or (ii) a domain capable of (i.e.suitable for) facilitating the introduction of a click chemistrymoiety(ies) configured to interact with another click chemistrymoiety(ies) on the capture domain of the capture probes of theinvention.

Thus, in some embodiments, step (d) may be viewed as fragmenting the DNAof the biological specimen and providing the DNA fragments with a domaincapable of (i.e. suitable for) facilitating the introduction of abinding domain capable of binding (hybridizing) to the capture domain ofthe capture probes of the invention.

In a representative embodiment, the adaptor of the transposome maycomprise a domain with a nucleotide sequence that templates the ligationof a universal adaptor to the tagmented DNA. The universal adaptorcomprises a binding domain capable of binding (hybridizing) to thecapture domain of the capture probes of the invention. Thus, in someembodiments, tagmentation provides the DNA of the biological specimenwith a binding domain indirectly.

In another representative embodiment, the adaptor of the transposome maycomprise a domain with a nucleotide sequence that is a substrate in aligation reaction that introduces a universal adaptor to the tagmentedDNA, e.g. a domain to which a universal adaptor may bind. For instance,the universal adaptor may be a partially double-stranded oligonucleotidehaving a first strand comprising a single-stranded portion containingdomain that binds to the adaptor sequence ligated to the fragmented(i.e. tagmented) DNA and a second strand comprising a domain that bindsto the first strand and a domain capable of binding (hybridizing) to thecapture domain of the capture probes of the invention. Ligation of theuniversal adaptor to the fragmented (i.e. tagmented) DNA provides thetagmented DNA with a domain that binds to the capture domain of thecapture probes of the invention. Thus, in some embodiments, tagmentationprovides the DNA of the biological specimen with a binding domainindirectly.

As tagmentation results in DNA that comprises gaps between the 3′ endsof the DNA of the biological specimen and the 5′ ends at the doublestranded portion of the adaptors (i.e. the 5′ ends of the adaptorscontaining the MEs are not ligated to the 3′ ends of the fragmented DNAof the biological specimen), providing the tagmented DNA with a bindingdomain capable of binding (hybridizing) to the capture domain of thecapture probes of the invention may require a step of “gap filling” thetagmented DNA.

Gap filling may be achieved using a suitable polymerase enzyme, i.e. aDNA polymerase (e.g. selected from the list below). In this respect, the3′ ends of the tagmented DNA are extended using the complementarystrands of the tagmented DNA as templates. Once the gaps have beenfilled, the 3′ ends of the tagmented DNA are joined to the 5′ ends ofthe adaptors by a ligation step, using a suitable ligase enzyme (e.g.selected from the list below).

It will be understood in this regard that the 5′ end of adaptorscontaining the ME is phosphorylated to enable ligation to take place.The transposome may comprise an adaptor in which one or both 5′ ends arephosphorylated. In embodiments where the transposome comprises anadaptor in which the 5′ end of adaptor containing the ME is notphosphorylated, the gap filling process may comprise a further step ofphosphorylating the 5′ end of the adaptor, e.g. using a kinase enzyme,such as T4 polynucleotide kinase.

In some embodiments, the 3′ ends of the tagmented DNA may be extendedusing a DNA polymerase with strand displacement activity using thecomplementary strands of the tagmented DNA as templates. This results inthe displacement of the strands of the adaptors that are not ligated tothe fragmented DNA and the generation of fully double stranded DNAmolecules. These molecules may be provided with a domain capable ofbinding to the capture domain of the capture probes by any suitablemeans, e.g. ligation of adaptors, “tailing” with a terminal transferaseenzyme etc.

Thus, in some embodiments, the method comprises a step of extending the3′ ends of the fragmented (i.e. tagmented) DNA using a polymerase withstrand displacement activity to produce fully double stranded DNAmolecules.

In some embodiments, the fully double stranded DNA molecules may beprovided with a binding domain capable of binding to the capture domainof the capture probes. In some embodiments, a binding domain may beprovided by ligation of adaptors to the double stranded DNA molecules orvia the use of a terminal transferase active enzyme to incorporate apolynucleotide tail, e.g. homopolymeric sequence (e.g. a poly-A tail),at the 3′ ends of the double stranded DNA molecules.

Thus, in preferred embodiments, step (d) results, directly orindirectly, in a biological specimen containing fragmented DNA (i.e.tagmented DNA) comprising a domain that binds to the capture domain ofthe capture probes of the invention. It will be evident from thedisclosures in WO 2012/140224 (herein incorporated by reference) thatthe fragmented DNA may be spatially tagged using various means,according to step (f). Representative embodiments of step (f) aredescribed in more detail below.

A “transposase” is an enzyme that binds to the end of a transposon andcatalyzes its movement to another part of the genome by a cut and pastemechanism or a replicative transposition mechanism.

Transposase Tn5 is a member of the RNase superfamily of proteins. TheTn5 transposon is a composite transposon in which two near-identicalinsertion sequences (IS50L and IS50R) flank three antibiotic resistancegenes. Each IS50 contains two inverted 19-bp end sequences (ESs), anoutside end (OE) and an inside end (IE).

A hyperactive variant of the Tn5 transposase is capable of mediating thefragmentation of double-stranded DNA and ligation of syntheticoligonucleotides (adaptors) at both 5′ ends of the DNA in a reactionthat takes about 5 minutes. However, as wild-type end sequences have arelatively low activity, they are preferably replaced in vitro byhyperactive mosaic end (ME) sequences. A complex of the Tn5 transposasewith 19-bp ME is thus all that is necessary for transposition to occur,provided that the intervening DNA is long enough to bring two of thesesequences close together to form an active Tn5 transposase homodimer.

Methods, compositions, and kits for treating nucleic acid, and inparticular, methods and compositions for fragmenting and tagging DNAusing transposon compositions are described in detail in US2010/0120098and US2011/0287435, which are hereby incorporated by reference in theirentireties.

Thus, any transposase enzyme with tagmentation activity, i.e. capable offragmenting DNA and ligating oligonucleotides to the ends of thefragmented DNA, may be used in the methods of the present invention. Insome embodiments, the transposase is a Tn5 or Mu transposase or afunctional variant or derivative thereof.

Thus, in some embodiments, the transposase, e.g. Tn5 or Mu or functionalvariant or derivative thereof, comprises an amino acid sequence with atleast 80% sequence identity to a sequence as set forth in SEQ ID NOs: 1or 2. In some embodiments, the functional variant or derivative is ahyperactive variant or derivative, i.e. a variant or derivative withincreased transposase activity relative to the naturally-occurringprotein.

Preferably said polypeptide sequence is at least 90, 91, 92, 93, 94, 95,96, 97, 98 or 99% identical to the sequence to which it is compared.

Sequence identity of polypeptide molecules may be determined by, e.g.using the SWISS-PROT protein sequence databank using FASTA pep-cmp witha variable pamfactor, and gap creation penalty set at 12.0 and gapextension penalty set at 4.0, and a window of 2 amino acids. Preferablysaid comparison is made over the full length of the sequence, but may bemade over a smaller window of comparison, e.g. less than 600, 500, 400,300, 200, 100 or 50 contiguous amino acids.

Preferably such sequence identity related polypeptides are functionallyequivalent to the one of the polypeptides set forth in SEQ ID NOs: 1 or2. As such, the polypeptides with a sequence as set forth in SEQ ID NOs:1 or 2 may be modified without affecting the sequence of thepolypeptide.

Modifications that do not affect the sequence of the polypeptideinclude, e.g. chemical modification, including by deglycosylation orglycosylation. Such polypeptides may be prepared bypost-synthesis/isolation modification of the polypeptide withoutaffecting functionality, e.g. certain glycosylation, methylation etc. ofparticular residues.

As referred to herein, to achieve “functional equivalence” thepolypeptide may show some increased or reduced efficacy in transposase(e.g. tagmentation) activity relative to the parent molecule (i.e. themolecule from which it was derived, e.g. by amino acid substitution),but preferably is as efficient or is more efficient. Thus, functionalequivalence relates to a polypeptide which has transposase activitycapable of fragmenting DNA and ligating oligonucleotides to the DNAfragments. This may be tested by comparison of the transposase activityof the derivative polypeptide relative to the polypeptide from which itis derived in a quantitative manner. The derivative is preferably atleast 30, 50, 70 or 90% as effective as the parent polypeptide in themethods of the invention. As noted above, in some preferred embodiments,the polypeptide is hyperactive relative to the parent polypeptideexemplified above, i.e. is at least about 110, 120, 130, 140, 150, 200,250 or 300% as effective as the parent polypeptide in the methods of theinvention.

Functionally-equivalent proteins which are related to or derived fromthe naturally-occurring protein, may be obtained by modifying the nativeamino acid sequence by single or multiple amino acid substitution,addition and/or deletion (providing they satisfy the above-mentionedsequence identity requirements), but without destroying the molecule’sfunction. Preferably the native sequence has less than 20 substitutions,additions or deletions, e.g. less than 10, 5, 4, 3, 2, or 1 suchmodifications. Such proteins are encoded by “functionally-equivalentnucleic acid molecules” which are generated by appropriate substitution,addition and/or deletion of one or more bases. As noted above, theinventors have determined that typical detergent-based permeabilizationconditions are not sufficient to enable a transposase (e.g. atransposome) to access its substrate, i.e. DNA (e.g. genomic DNA), whenthe biological specimen (e.g. tissue section) is immobilized on a solidsubstrate, e.g. array. Accordingly, the step of “permeabilizing thebiological specimen under conditions sufficient to make DNA in thebiological specimen accessible to a transposase enzyme” refers to theuse of any conditions that enable a transposase to access its substrate,i.e. DNA (e.g. genomic DNA), when the biological specimen (e.g. tissuesection) is immobilized on a solid substrate, e.g. array.

It will be evident that biological specimens, e.g. tissue samples, fromdifferent sources may require different treatments to make themaccessible to the transposase (i.e. to enable the transposase to accessand act on its substrate). If the tissue sample is not permeabilizedsufficiently the transposase will not interact with the DNA of thebiological specimen and the amount of tagmentation may be too low toenable further analysis. Conversely, if the biological specimen, e.g.tissue sample, is too permeable, tagmented DNA (and other nucleic acids,e.g. RNA) may diffuse away from its origin in the biological specimen,e.g. tissue sample, i.e. the tagments (and other nucleic acids, e.g.RNA) captured by the capture probes may not correlate accurately withtheir original spatial distribution in the biological specimen, e.g.tissue sample. Hence, there must be a balance between permeabilizing thebiological specimen, e.g. tissue sample, enough to obtain enableefficient interaction between the transposase and DNA whilst maintainingthe spatial resolution of the nucleic acid distribution in thebiological specimen, e.g. tissue sample.

Thus, the permeabilization conditions in step (c) may be adapted to thecharacteristics of the biological specimen. For instance, the enzyme(s)and/or chemicals (e.g. buffer(s)) used in step (c) may be selectedaccording to the tissue type.

Moreover, the inventors have determined that the permeabilizationconditions in step (c) may be adapted to enable uniform DNAfragmentation to enable capture of DNA tagments regardless of chromatinaccessibility or to yield fragments with a pronounced nucleosomalpattern. Thus, the permeabilization conditions in step (c) may beselected according to the level of fragmentation required or the DNAmolecules of interest, i.e. the DNA molecules to be spatially taggedaccording to the methods of the invention.

Representative permeabilization conditions are described below. It willbe evident that these representative conditions may be modified oradapted to suit the biological specimen, transposase and DNAfragmentation, and such modifications are within the purview of theskilled person.

The permeabilization conditions in step (c) may comprise subjecting thebiological specimen to chemical and/or enzymatic permeabilizationconditions.

In some embodiments, the chemical permeabilization conditions comprisecontacting the biological specimen with an alkaline solution, e.g. abuffered solution with a pH of about 8.0-11.0, such as about 8.5-10.5 orabout 9.0-10.0, e.g. about 9.5. In some embodiments, the buffer is aglycine-KOH buffer.

As shown in the Examples, the inventors have found that permeabilizationmay be performed using pepsin. Notably, the level of DNA fragmentationupon treatment with a transposase can be controlled by changing thepepsin permeabilization conditions. For instance, permeabilization usingpepsin in the presence of 100 mM HCl (i.e. having a pH of about 1.0)induces uniform DNA fragmentation and may be used to capture DNAtagments regardless of chromatin accessibility. Alternatively,permeabilization using pepsin in the presence of 0.5 M acetic acid (i.e.having a pH of about 2.5) provides partial recovery of the nucleosomalpattern typically associated with accessible chromatin.

Thus, in some embodiments, the permeabilization conditions in step (c)may comprise contacting the biological specimen with an acidic solutioncomprising a protease enzyme.

In some embodiments, the permeabilization conditions in step (c) maycomprise contacting the biological specimen with a reaction mixture(e.g. solution) comprising an aspartyl protease (e.g. pepsin) in anacidic buffer, e.g. a buffer with a pH of about 4.0 or less, such asabout 3.0 or less, e.g. about 0.5-3.0 or about 1.0-2.5.

In a preferred embodiment, the aspartyl protease is a pepsin enzyme,pepsin-like enzyme or a functional equivalent thereof. Thus, any enzymeor combination of enzymes in the enzyme commission number 3.4.23.1 maybe used in the present invention.

Thus, in some embodiments, the pepsin enzyme is selected from thefollowing group, which refers to the UniProtKB/Swiss-Prot accessionnumbers: P03954/PEPA1_MACFU; P28712/PEPA1_RABIT; P27677/PEPA2_MACFU;P27821/PEPA2_RABIT; P0DJD8/PEPA3_HUMAN; P27822/PEPA3_RABIT;P0DJD7/PEPA4_HUMAN; P27678/PEPA4_MACFU; P28713/PEPA4_RABIT;P0DJD9/PEPA5_HUMAN; Q9D106/PEPA5_MOUSE; P27823/PEPAF_RABIT;P00792/PEPA_BOVIN; QIN2D4/PEPA_CALJA; Q9GMY6/PEPA_CANLF;P00793/PEPA_CHICK; P11489/PEPA_MACMU; P00791/PEPA_PIG;Q9GMY7/PEPA_RHIFE; Q9GMY8/PEPA_SORUN; P81497/PEPA_SUNMU;P13636/PEPA_URSTH and functional variants and derivatives thereof or acombination thereof.

In some embodiments, the pepsin enzyme is selected from following group,which refers to the UniProtKB/Swiss-Prot accession numbers:P00791/PEPA_PIG; P00792/PEPA_BOVIN and functional variants andderivatives thereof or a combination thereof.

By a “functional variant or derivative” is meant that a mutant ormodified protease (i.e. containing one or more amino acid substitutions,deletions or additions relative to the protease from which is wasderived), which may show some reduced protease activity relative to theactivity of the protease from which it is derived in conditions that areoptimum for the enzyme, e.g. in the buffer, salt and temperatureconditions recommended by the manufacturer. Thus, a variant orderivative protease may be considered to be functional if it has atleast 50%, e.g. at least 60, 70, 80, 85, 90, 95, 96, 97, 98, 99 or 100%,activity relative to the activity of the protease from which it wasderived in conditions that are optimum for the enzyme.

Thus, in some embodiments, the pepsin enzyme or functional variant orderivative thereof, comprises an amino acid sequence with at least 80%sequence identity to a sequence as set forth in SEQ ID NOs: 3 or 4.

Preferably said polypeptide sequence is at least 90, 91, 92, 93, 94, 95,96, 97, 98 or 99% identical to the sequence to which it is compared.

The inventors have alternatively found that permeabilization may beperformed using collagenase, which provides efficient genomeaccessibility to the transposase while preserving nuclear integrity.Notably, permeabilization with collagenase yields pronounced nucleosomalpattern that is typically associated with chromatin tagmentation.Collagenases are zinc endopeptidases and are typically inhibited by bothEDTA and EGTA. Collagenases may be isolated from Clostridiumhistolyticum.

Thus, in some preferred embodiments, step (c) comprises contacting thebiological specimen with a zinc endopeptidase (e.g. collagenase) underconditions suitable for proteolytic (e.g. collagenase) activity, e.g. ina buffered solution with a pH of about 7.0-8.0, e.g. about 7.4.

Thus, in some embodiments, the biological specimen is contacted with azinc endopeptidase (e.g. collagenase) in the absence of a chelator ofdivalent cations, such as EDTA or EGTA. In some embodiments, it may beuseful to stop the zinc endopeptidase (e.g. collagenase)permeabilization step by contacting the biological specimen with achelator of divalent cations, such as EDTA or EGTA.

In a preferred embodiment, the zinc endopeptidase is a collagenaseenzyme, collagenase-like enzyme or a functional equivalent thereof.Thus, any enzyme or combination of enzymes in the enzyme commissionnumber 3.4.23.3 may be used in the present invention.

Thus, in some embodiments, the collagenase is selected from thefollowing group, which refers to the UniProtKB/Swiss-Prot accessionnumbers: P43153/COLA_CLOPE; P43154/COLA_VIBAL; Q9KRJ0/COLA_VIBCH;Q56696/COLA_VIBPA; Q8D4Y9/COLA_VIBVU; Q9X721/COLG_HATHI;Q46085/COLH_HATHI; Q899Y1/COLT_CLOTE URSTH and functional variants andderivatives thereof (defined above) or a combination thereof.

In some embodiments, the pepsin enzyme is selected from following group,which refers to the UniProtKB/Swiss-Prot accession numbers:Q9X721/COLG_HATHI; Q46085/COLH_HATHI and functional variants andderivatives thereof or a combination thereof.

Thus, in some embodiments, the collagenase enzyme or functional variantor derivative thereof, comprises an amino acid sequence with at least80% sequence identity to a sequence as set forth in SEQ ID NOs: 5 or 6.

Preferably said polypeptide sequence is at least 90, 91, 92, 93, 94, 95,96, 97, 98 or 99% identical to the sequence to which it is compared.

The inventors have also found that permeabilization may be performedusing proteinase K, which allows recovery of unprotected DNA tagments,i.e. permeabilization with proteinase K may be used to capture DNAtagments regardless of chromatin accessibility.

Thus, in some preferred embodiments, step (c) comprises contacting thebiological specimen with a serine protease (e.g. proteinase K) underconditions suitable for proteolytic (e.g. proteinase K) activity.Advantageously, the serine protease (e.g. proteinase K) is active over awide pH range (e.g. from about 6.5 and 9.5), under denaturing conditions(e.g., in the presence of SDS or urea), in the presence of metalchelating agents (e.g., EDTA) and at comparatively high temperatures(e.g. about 45° C. to about 65° C.).

In a preferred embodiment, the serine protease is a proteinase K enzyme,proteinase K-like enzyme or a functional equivalent thereof. Thus, anyenzyme or combination of enzymes in the enzyme commission number3.4.21.64 may be used in the present invention.

Thus, in some embodiments, the proteinase K is P06873/PRTK_PARAQ, whichrefers to the UniProtKB/Swiss-Prot accession numbers, or a functionalvariant or derivative thereof (defined above) or a combination thereof.

Thus, in some embodiments, the proteinase K enzyme or functional variantor derivative thereof, comprises an amino acid sequence with at least80% sequence identity to a sequence as set forth in SEQ ID NO: 7.

Preferably said polypeptide sequence is at least 90, 91, 92, 93, 94, 95,96, 97, 98 or 99% identical to the sequence to which it is compared.

Commercially available proteases are commonly isolated from theirnative, e.g. animal or microbial source. However, the proteases may beproduced recombinantly, e.g. from a microbial, e.g. bacterial,expression system. The source of the protease for use in the presentinvention is not particularly important and both natural and recombinantproteases are contemplated for use in the methods described herein.

The step of permeabilizing the biological specimen using the chemicaland/or enzymatic reagents defined above may be performed under anysuitable conditions, e.g. concentration, time, temperature etc. whichmay be adapted based on the origin of the biological specimen (e.g. theorganism and/or organ from which the biological specimen was obtained)and the chemical and/or enzymatic reagents.

In some embodiments, the protease enzymes may be used at a concentrationof about 0.05 mg/ml to about 1 mg/ml, e.g. about 0.1 mg/ml to about 0.5mg/ml.

In some embodiments, the biological specimen may be incubated with theprotease enzymes and/or chemical reagents (e.g. alkaline buffer) forabout 1-5 minutes, e.g. about 1, 2, 3, 4, 5 minutes. For instance, thepepsin and proteinase K enzymes (or functional equivalents etc.) may beincubated with the biological specimen for about 2-4 minutes, e.g. about3 minutes. It will be evident that the incubation period may depend onthe concentration of the enzyme and the conditions of use, e.g. buffer,temperature etc. Thus, in some embodiments, the protease enzymes may beincubated with the biological specimen for more or less time than theperiods set out above. Such modifications are within the purview of theskilled person.

Thus, in some embodiments, the biological specimen may be incubated withthe protease enzymes and/or chemical reagents (e.g. alkaline buffer) forat least about 5 minutes, e.g. at least about 10, 12, 15, 18 or 20minutes. For instance, the collagenase enzymes (or functionalequivalents etc.) may be incubated with the biological specimen forabout 10-30 minutes, e.g. about 20 minutes.

The permeabilization step may be stopped (e.g. the protease activity maybe stopped) by any suitable means. For instance, the reaction mixture(e.g. solution) comprising the protease enzymes and/or chemical reagentsmay be removed from the solid substrate (e.g. array), i.e. separatedfrom the biological specimen. Alternatively or additionally, theprotease enzyme(s) may be inhibited (e.g. by the addition of aninhibitor, such as EDTA for collagenase) or denatured (e.g. by theaddition of a denaturing agent or increasing the temperature).

Representative temperature conditions for the permeabilization stepinclude incubation at about 10-70° C. depending on the enzyme. Forinstance, pepsin and collagenase may be used at about 10-44, 11-43,12-42, 13-41, 14-40, 15-39, 16-38, 17-37° C., e.g. about 10, 12, 15, 18,20, 22, 25, 28, 30, 33, 35 or 37° C., preferably about 30-40° C., e.g.about 37° C. Proteinase K may be used at about 40-70° C., e.g. about50-70, 60-70 e.g. about 65° C.

In some embodiments, the reaction mixture (e.g. solution) comprising theproteases defined above may contain other components, e.g. buffer, salt,etc. sufficient to ensure that the proteases are functional. Forinstance, in some embodiments, the reaction mixture further comprises analbumin protein, such as BSA. In some preferred embodiments, thereaction mixture (e.g. solution) comprising the collagenase enzyme (orfunctional variant or derivative thereof) comprises an albumin protein,such as BSA.

The step of fragmenting the DNA in the biological specimen comprisescontacting the biological specimen containing DNA with the transposase,e.g. transposome, i.e. a reaction mixture (e.g. solution) comprising atransposase, e.g. transposome, as defined herein under any suitableconditions, i.e. conditions that result in the fragmentation (e.g.tagmentation) of said biological specimen. Typical conditions willdepend on the transposase used and may be determined using routinemethods known in the art. Thus, alternatively viewed, suitableconditions may be conditions (e.g. buffer, salt, temperature conditions)under which the transposase is functional, e.g. displays transposaseactivity, particularly tagmentation activity in the biological specimen.

By “functional” is meant that the transposase may show some reducedactivity relative to the activity of the transposase in conditions thatare optimum for the enzyme, e.g. in the buffer, salt and temperatureconditions recommended by the manufacturer. Thus, the transposase may beconsidered to be functional if it has at least 50%, e.g. at least 60,70, 80, 85, 90, 95, 96, 97, 98, 99 or 100%, activity relative to theactivity of the transposase in conditions that are optimum for theenzyme.

In some embodiments, the reaction mixture (solution) comprising thetransposase may contain other components, e.g. buffer, salt, etc.sufficient to ensure that the transposase is functional. For instance,in some embodiments, the reaction mixture further comprises spermidine.

In a representative example, the reaction mixture comprises atransposase enzyme in a buffered solution (e.g. Tris-acetate) having apH of about 6.5-8.5, e.g. about 7.0-8.0 such as about 7.5. Additionallyor alternatively, the reaction mixture may be used at any suitabletemperature, such as about 10-45° C., e.g. about 10-44, 11-43, 12-42,13-41, 14-40, 15-39, 16-38, 17-37° C., e.g. about 10, 12, 15, 18, 20,22, 25, 28, 30, 33, 35 or 37° C., preferably about 30-40° C., e.g. about37° C.

The “adaptors” or “oligonucleotides” in the complex with the transposase(i.e. that form part of the transposome, MEDS as described above)comprise partially double stranded oligonucleotides. The double strandedportion of the adaptors contains Mosaic End (ME) sequences. The singlestranded portion of the adaptors (5′ overhang) contains the functionaldomain or sequence to be incorporated in the fragmented (i.e. tagmented)DNA. Thus, the functional domain is on the strand of the adaptor thatwill be ligated to the fragmented DNA. In other words, the functionaldomain is located upstream (i.e. 5′ to) the ME sequence, i.e. in the 5′overhang of the adaptor.

As noted above, in some embodiments, the functional domain may be adomain that binds to the capture domain of the capture probes of theinvention.

In some embodiments, the functional domain may be a domain thatfacilitates the introduction of a binding domain that binds to thecapture domain of the capture probes of the invention, i.e. a domainthat hybridises to a universal adaptor or templates the ligation of auniversal adaptor to the tagmented DNA.

In some embodiments, the ME sequence is a Tn5 transposase recognitionsequence (e.g. as set forth in SEQ ID NO: 8). In some embodiments, theME sequence is a Mu transposase recognition sequence (e.g. as set forthin any one of SEQ ID NOs: 9-14).

Thus, in a further aspect, the invention may be seen as providing acomposition comprising a transposase enzyme (e.g. as defined herein)complexed with an adaptor comprising transposon end sequences (or mosaicends as defined herein) and a domain that binds to a capture probe asdefined herein (e.g. a homopolymeric sequence) for use in a method forspatially tagging nucleic acids of a biological specimen, such as themethods defined herein.

A transposome may be produced by loading a transposase enzyme (e.g. apurified enzyme) with the adaptors described above. It will be evidentfrom the representative embodiments described herein that the singlestranded portion of the adaptor of the transposome may require aphosphorylated 5′ end, e.g. to enable ligation of tagmented DNA to thecapture probes.

Thus, in some embodiments, the transposase used in step (d) (or in thecomposition defined above) is in the form of a transposome comprising anadaptor (MEDS) in which the 5′ overhang is phosphorylated.

Whilst the adaptors may be phosphorylated prior to their assembly withthe transposase to form the transposome, in-solution assembly of thetransposome is inefficient. In this respect, the inventors havedetermined that phosphorylation of adaptors when complexed with atransposase (i.e. phosphorylation in situ in the transposome) results inimproved tagmentation, e.g. relative to a transposome produced byin-solution assembly with adaptors (MEDS) with phosphorylated 5′overhangs.

As described in the Examples, transposomes comprise the adaptors (MEDS)described above (i.e. comprising 5′ overhangs). If the 5′ overhang ofthe adaptor is not phosphorylated prior to its assembly in thetransposome, it will have accessible 5′ hydroxyl groups outside of themosaic-end transposase binding site. Thus, phosphorylation of the 5′overhang of the assembled transposome complexes may be achieved byexposing these 5′ ends of transposome complexes to a polynucleotidekinase (e.g. T4-polynucleotide kinase (T4-PNK)) in the presence of ATP.

Thus, in some embodiments, step (d) comprises fragmenting DNA of thebiological specimen with a transposome as defined herein and maycomprise a further step of phosphorylating the 5′ ends of the adaptors(particularly the 5′ overhangs of the adaptors, i.e. MEDS) in thetransposome complex.

Alternatively viewed, in some embodiments, the method comprises a stepof providing a transposome that has been treated to phosphorylate the 5′ends of the adaptors (particularly the 5′ overhangs of the adaptors,i.e. MEDS) in the transposome complex, i.e. step (d) comprisesfragmenting the biological specimen with a transposome that has beentreated to phosphorylate the 5′ ends of the adaptors (particularly the5′ overhangs of the adaptors, i.e. MEDS) in the transposome complex.

Any suitable enzyme and conditions may be used to phosphorylate the 5′ends of the adaptors (particularly the 5′ overhangs of the adaptors,i.e. MEDS) in the transposome complex, e.g. T4-PNK or T7-PNK. In arepresentative embodiment, the phosphorylation reaction may be carriedout by contacting the transposome with a polynucleotide kinase (e.g.T4-PNK or T7-PNK) in a buffered solution (e.g. Tris-HCl, pH about7.0-8.0, e.g. about 7.6) at about 20-40° C., e.g. about 25-37° C., forabout 1-60 minutes, e.g. about 5-50, 10-40, 20-30 minutes.

In some embodiments, the step (d) comprises the formation of a pluralityof transposase-DNA fragment complexes, wherein a transposase-DNAfragment complex of the plurality of transposase-DNA fragment complexescomprises a DNA fragment. In an additional embodiment, prior to step (e)the plurality of transposase-DNA fragment complexes is treated todissociate a transposase from a transposase-DNA fragment complex of theplurality of transposase-DNA fragment complexes. In one otherembodiment, a DNA fragment is released from the dissociated transposase.In one embodiment, the dissociation of the transposase from a DNAfragment is achieved by contacting the transposase-DNA fragment complexwith a stimulus. In other embodiments, the stimulus may be a chemicalstimulus (e.g., EDTA) or a temperature stimulus.

In one embodiment, the fragmented DNA of (d) is subjected to one or morenucleic acid reactions. In one other embodiment, prior to (e) thefragment the fragmented DNA of (c) is subjected to one or more nucleicacid reactions. In other embodiments, the one or more nucleic acidreactions comprise a nucleic acid amplification and/or a nucleic acidmodification. In another embodiment, the nucleic acid amplification isby an RNA polymerase or a DNA polymerase.

Step (f)(i) in the method above may involve extending the capture probesusing the nucleic acid molecules hybridised to the capture probes (i.e.“captured” by the capture probes) as extension templates to produceextended probes thereby spatially tagging the nucleic acids (e.g.tagments) of the biological specimen.

In the context of DNA, step (f)(i) may be viewed as generating DNA(particularly tagged DNA) from the captured DNA, e.g. relating to thesynthesis of a complementary strand of DNA. This may involve a step ofDNA polymerisation, extending the capture probe, which functions as theprimer, using the captured DNA (e.g. tagments) as a template to producea complementary strand of the DNA hybridized to the capture probe.

As described above, step (d) of the method involves providing thefragmented DNA with a domain that binds to the capture domain in thecapture probe, directly or indirectly. Thus, in embodiments of step(f)(i) where the capture probes are extended using the DNA hybridized tothe capture probes as extension templates, the domain that binds to thecapture domain in the capture probes is provided at the 3′ end of thefragmented (i.e. tagmented) DNA (see e.g. FIG. 13 ). As tagmentationresults in the ligation of adaptor sequences to the 5′ ends of thefragmented DNA, in this embodiment a domain that binds to the capturedomain in the capture probes must be provided indirectly.

In some embodiments, the domain that binds to the capture domain in thecapture probes forms a single stranded domain at the 3′ end of thetagmented DNA, i.e. a 3′ overhang, such as a homopolymeric sequence(e.g. poly-A sequence). Thus, the 3′ overhang binds to the capturedomain of the capture probes (step (e)) and the bound DNA strandtemplates the extension of the capture probe via a polymerizationreaction. If the DNA hybridized to the capture probes is partiallydouble stranded, the extension reaction may use a DNA polymerase withstrand displacement activity as described below.

In some embodiments, it may be advantageous or necessary to make thetagmented DNA single-stranded, e.g. where the domain that binds to thecapture domain in the capture probes does not form a 3′ overhang. Forinstance, the domain that binds to the capture domain in the captureprobes may be formed by extending the 3′ end of the tagmented DNA togenerate a sequence that is complementary to the functional domain inthe adaptor ligated to the tagmented DNA. In a representativeembodiment, the functional domain of the adaptor ligated to the DNA maycomprise a homopolymeric sequence (e.g. a poly-T sequence) and extendingthe 3′ end of the tagmented DNA results in the production of acomplementary homopolymeric sequence (e.g. a poly-A sequence) that bindsto the capture domain of the capture probes. Thus, in some embodiments,step (e) may comprise a step of making the tagmented DNAsingle-stranded, e.g. denaturing the DNA. Suitable methods forgenerating single-stranded DNA are known in the art, e.g. heat.

Other embodiments of step (f)(i) in the method above may involveextending the capture probes using the nucleic acid molecules (e.g.tagments) hybridised to the capture probes (i.e. “captured” by thecapture probes) as ligation templates to produce extended probes therebyspatially tagging the nucleic acids of the biological specimen.

Thus, in the context of DNA, step (f)(i) may be viewed as generating DNA(particularly tagged DNA) from the captured DNA relating to the ligationof the DNA. This may involve a step of DNA ligation, extending thecapture probe, which is ligated to the complementary strand of the DNAhybridized to the capture probe using the captured DNA as a ligationtemplate.

It will be evident that the way in which the tagmented DNA is ligated tothe capture probe will depend on the orientation of the capture probe onthe array, e.g. whether it is immobilized via its 3′ end or 5′ end, andwhether the capture probe is immobilized on the solid substrate (e.g.array) directly or indirectly (e.g. via a hybridization to anoligonucleotide that is directly immobilized on the array, e.g. asurface probe).

Whilst it is contemplated that the capture probes of the invention maybe immobilized via their 3′ ends, such that they have a free 5′ end thatcan be ligated to the tagmented DNA, it is preferred that the captureprobes are immobilized via their 5′ ends, i.e. such that they have afree 3′ end that can participate in a ligation or extension reaction.

Thus, in a representative embodiment of step (f)(i), the tagmented DNAis provided with a domain that binds to the capture domain in thecapture probes at the 3′ end of the fragmented (i.e. tagmented) DNA asdescribed above, i.e. a 3′ overhang, such as a homopolymeric sequence(e.g. poly-A sequence). Thus, the 3′ overhang binds to the capturedomain of the capture probes (step (e)) and the bound DNA strandtemplates the ligation of the capture probe to the strand that iscomplementary to the bound DNA strand. As described above, it ispreferred that the adaptor of the transposome (i.e. the functionaldomain of the adaptor) contains a phosphorylated 5′ end to enableligation of tagmented DNA to the capture probes. However, in someembodiments, the adaptors may not contain phosphorylated 5′ ends andthus the tagmented DNA may be phosphorylated after step (d).

Step (f)(ii) may be viewed as generating DNA (particularly tagged DNA)from the captured DNA involving a step of DNA ligation, extending thecapture probe, which is ligated to the strand of the DNA hybridized tothe capture probe using the capture probe as a ligation template.

It will be evident that the way in which the tagmented DNA is ligated tothe capture probe will depend on the orientation of the capture probe onthe array, e.g. whether it is immobilized via its 3′ end or 5′ end, andwhether the capture probe is immobilized on the directly or indirectly(e.g. via a hybridization to an oligonucleotide that is directlyimmobilized on the array, e.g. a surface probe).

In some embodiments, the capture probes may be immobilized indirectly onthe array via hybridization to so-called surface probes. Thus, in someembodiments, the capture probes may be viewed as partiallydouble-stranded probes, wherein at least the capture domain of thecapture probe is single stranded.

Thus, in a representative embodiment, the capture probes are partiallydouble-stranded probes containing a first strand comprising a capturedomain and positional domain (a “capture domain oligonucleotide”) and asecond strand (a “surface probe”) comprising a sequence that iscomplementary to the positional domain, wherein the positional domainand sequence that is complementary to the positional domain form thedouble stranded portion of the capture probe. The second strand mayfurther comprise an amplification domain and/or cleavage domain asdescribed below. Thus, the second strand of the partiallydouble-stranded probe is a so-called surface probe.

In some embodiments, the surface probe (i.e. second strand of thecapture probe) is immobilized on the array via its 5′ end and tagmentedDNA is provided with a domain that binds to the capture domain of thecapture probe directly, i.e. the adaptor of the transposome comprises anME sequence and a nucleotide sequence (functional domain) that iscomplementary to the capture domain of the capture probes (i.e. thefirst strand of the partially double stranded capture probe).Accordingly, step (f)(ii) comprises a step extending the second strands(surface probes) of the partially double-stranded capture probes usingthe capture domain oligonucleotide as a ligation template to ligate thenucleic acids that hybridize to the capture domains of the captureprobes to the second strands (surface probes) of the partiallydouble-stranded capture probes thereby extending the capture probes (thesecond strands (surface probes) of the partially double-stranded captureprobes) to produce extended probes (i.e. probes that comprise thenucleic acids that hybridize to the capture domains of the captureprobes and sequences complementary to the positional domains of thecapture probes), thereby spatially tagging the nucleic acids of thebiological specimen.

It will be evident that the first strand of the partially doublestranded capture probes (the capture domain oligonucleotide) does notneed to be hybridized to the second strand (surface probe) during all ofthe steps of the method described herein. It is only necessary for thefirst strand to be present in steps (e) and (f) of the method, i.e. toenable the tagmented DNA to hybridise to the capture probes and totemplate the ligation reaction. Thus, in some embodiments, the methodmay comprise a further step of hybridizing a capture domainoligonucleotide to surface probes immobilized on the array. In someembodiments, this step occurs as part of step (e).

Whilst it is preferred that the first strand of the partially doublestranded capture probes contains the capture domain and the positionaldomain, such that the first and second strands of the partially doublestranded capture probes are hybridised via the positional domain, itwill be evident that this is not essential to spatially tag nucleicacids in the embodiment described above. In this respect, it may beadvantageous for the capture domain and positional domain to be providedon different strands of the partially double-stranded capture probes.For instance, the surface probes may comprise the positional domain anda domain that is complementary to a domain in the capture domainoligonucleotide. When the surface probes are immobilized via their 5′ends, the domain that binds to the capture domain oligonucleotide isdownstream (i.e. 3′ of) the domain of the positional domain. Thus, insome embodiments, the capture domain and positional domain are providedon separate strands of a partially double stranded capture probe. Thisembodiment is particularly advantageous when the first strand of thepartially double stranded capture probe (i.e. comprising the capturedomain, the “capture domain oligonucleotide”) is provided during step(e) as described above. For instance, the domain that forms the doublestranded portion of the first and second strands of the capture probesmay be common to all of the surface probes and capture domainoligonucleotides, such that the same capture domain oligonucleotidehybridizes to all of the surface probes to produce the partially doublestranded capture probes of the invention.

It will be understood that equivalent embodiments may be performed inwhich the “surface probes” are immobilized via their 3′ end. In theseembodiments, it may be necessary that the 5′ end of the second strand ofthe capture probe (surface probe) is phosphorylated to enable ligationto take place.

The method of the invention enables the capture of DNA and RNA from thesame biological specimen, e.g. simultaneous capture.

Thus, step (f)(i) in the method above will be seen as relating to usingDNA or both DNA and RNA hybridized to the capture probes as extensiontemplates to produce extended probes. In some embodiments, step (f)(i)may involve using only tagmented DNA as extension templates to produceextended capture probes, i.e. step (f)(i) involves a DNA polymerasereaction to produce DNA. In some embodiments, step (f)(i) may involveusing both RNA and DNA as the extension templates to produce extendedcapture probes, i.e. step (f)(i) involves a reverse transcriptionreaction to produce cDNA and a DNA polymerase reaction to produce DNA.

In some embodiments, it may be desirable to perform separate extensionreactions for each type of nucleic acid to be detected. For instance, itis well-known in the art that RNA is less stable than DNA. Thus, in someembodiments, step (f)(i) may comprise a first extension reaction, whichis a reverse transcription reaction (to produce first strand of cDNA)followed by a second extension reaction which is a DNA polymerasereaction (to produce a DNA strand that is complementary to the DNAstrand hybridized to the probe). In some embodiments, the firstextension reaction is a DNA polymerase reaction and the second extensionreaction is a reverse transcription reaction.

In some embodiments, it may be desired to capture RNA via an extensionreaction and DNA via a ligation reaction. For instance, in someembodiments, step (f) may comprise an extension reaction, which is areverse transcription reaction (to produce first strand of cDNA)followed by a ligation reaction. Thus, in some embodiments, the methodcomprises spatially tagging DNA (e.g. gDNA) by ligating the DNAfragments to the surface probes and spatially tagging RNA by producingextended probes comprising cDNA as described below.

As described above the method may involve a step of providing the DNAfragments with a binding domain capable of hybridizing to the capturedomain of the capture probe. In some embodiments, the binding domain isthe same domain used to hybridize RNA in the biological specimen to thecapture probes, e.g. a poly-A domain. In some embodiments, the capturedomain may be a random sequence, e.g. a random hexamer sequence.

In some embodiments, it may be advantageous to perform the extensionreactions simultaneously. For instance, the extension reactions may beperformed simultaneously by combining the means for achieving RNAtemplated extension of said capture probes (e.g. a reversetranscriptase) with the means for achieving DNA templated extension orligation of the capture probes (e.g. a DNA polymerase or DNA ligase).

It is established in the art that some reverse transcriptase enzymes(e.g. Avian Myeloblastosis Virus (AMV) Reverse Transcriptase and MoloneyMurine Leukemia Virus (M-MuLV, MMLV) Reverse Transcriptase) cansynthesize a complementary DNA strand using both RNA (cDNA synthesis)and single-stranded DNA (ssDNA) as a template. Thus, in someembodiments, the extension reaction may utilize an enzyme (reversetranscriptase) that is capable of using both RNA and ssDNA as thetemplate for an extension reaction, e.g. an AMV or MMLV reversetranscriptase. Simultaneous extension reactions does not necessarilymean that all capture probes will be extended at the same time, butrather that the means for extending the capture probes are applied tothe solid substrate, e.g. array, simultaneously, i.e. at substantiallythe same time.

The phrase “at the same time” means substantially the same time, i.e.one component may be contacted with the solid substrate before the othercomponent, e.g. within seconds, (e.g. within 15, 30, 45, 60, 90, 120 or180 seconds) or minutes (e.g. within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12or 15 minutes), but such that the reactions are allowed to proceedtogether. If one component is contacted with the solid substrate beforethe other component it is preferred that the means for achieving RNAtemplated extension of said capture probes (e.g. reverse transcriptase)is contacted first and the means for achieving DNA templated extensionof said capture probes (e.g. DNA polymerase or DNA ligase) is contactedwithin seconds or minutes as defined above. However, in someembodiments, it may be desirable to contact the means for achieving DNAtemplated extension of said capture probes first and contact the meansfor achieving RNA templated extension of said capture probes withinseconds or minutes as defined above.

In view of the fact that step (f) may comprise sequential extensionreactions, it will be evident that the sequential extension reactionsmay be achieved by contacting the solid substrate with the means forachieving RNA templated extension of said capture probes and means forachieving DNA templated extension or ligation of said capture probesseparately.

Thus, in some embodiments, step (f) may be seen to comprise contactingsaid solid substrate, e.g. array, with means for achieving RNA templatedextension of said capture probes and subsequently contacting said solidsubstrate, e.g. array, with means for achieving DNA templated extensionor ligation of said capture probes.

The term “subsequently” means that the means for achieving DNA templatedextension or ligation of said capture probes is contacted with the solidsubstrate after the means for achieving RNA templated extension of saidcapture probes is contacted with the solid substrate or vice versa.There is no particular limit on the amount of time that may be allowedto lapse between the first and second reactions. However, if the firstreaction comprises a DNA templated extension or ligation of said captureprobes it is preferred that the second reaction is performed (i.e. meansfor the RNA templated extension of said capture probes is contacted withthe solid substrate) before the RNA molecules have substantiallydegraded. Thus, in some embodiments, “subsequently” means performing thesecond reaction minutes or hours after the first extension reaction iscompleted. For instance, the second reaction may be performed at least1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50 or 60 minutesafter the first reaction is completed, e.g. within 120, 90 or 60minutes, i.e. between 1-120, 5-90, 10-60 minutes after the firstreaction is completed. In some embodiments, the second reaction may beperformed at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 18, 24, 36 or 48hours after the first reaction is completed, e.g. within 72, 48 or 24hours, i.e. between 1-72, 6-48, 12-24 hours after the first reaction iscompleted.

In some embodiments, the means for achieving RNA templated extension ofsaid capture probes (e.g. reverse transcriptase) and means for achievingDNA templated extension or ligation of said capture probes (e.g. DNApolymerase or DNA ligase) are combined in a single reaction mixture,which is contacted with the solid substrate (e.g. array), e.g. thereverse transcriptase and DNA polymerase activities are provided byseparate enzymes. Thus, in some embodiments, step (f) comprisescontacting said solid substrate (e.g. array) with a reaction mixturecomprising:

-   (i) a DNA polymerase enzyme capable of extending said capture probes    using DNA hybridised to the capture probes as extension templates or    a DNA ligase enzyme capable of extending said capture probes using    DNA hybridised to the capture probes or the capture probes as    ligation templates; and-   (ii) a reverse transcriptase enzyme capable of extending said    capture probes using RNA hybridised to the capture probes as    extension templates.

Accordingly, the invention can be seen to provide the use of a reactionmixture comprising:

-   (i) a DNA polymerase enzyme capable of extending said capture probes    using DNA hybridised to the capture probes as extension templates or    a DNA ligase enzyme capable of extending said capture probes using    DNA hybridised to the capture probes or the capture probes as    ligation templates; and-   (ii) a reverse transcriptase enzyme capable of extending said    capture probes using RNA hybridised to the capture probes as    extension templates,-   in a method for spatially tagging nucleic acids of a biological    specimen, such as the methods defined herein.

In embodiments where step (f) comprises the use of a reaction mixturecomprising a DNA polymerase enzyme or DNA ligase enzyme and a reversetranscriptase enzyme the enzymes must be functional in the sameconditions, e.g. functional in the same buffer, salt, temperatureconditions.

By “functional” is meant that the enzymes may show some reducedpolymerase or ligase activity (target templated extension or ligation)relative to the activity in conditions that are optimum for the enzymes,e.g. in the buffer, salt and temperature conditions recommended by themanufacturer. Thus, the enzymes may be considered to be functional ifthey have at least 50%, e.g. at least 60, 70, 80, 85, 90, 95, 96, 97,98, 99 or 100%, activity relative to the activity of the polymerases inconditions that are optimum for the enzyme.

As noted above, In some embodiments, the means for achieving RNAtemplated extension of said capture probes (e.g. reverse transcriptase)and means for achieving DNA templated extension of said capture probes(e.g. DNA polymerase) are provided by a single enzyme that is capable ofusing both RNA and ssDNA as the template for an extension reaction, e.g.an AMV or MMLV reverse transcriptase.

The method of the invention may be used to capture (i.e. spatially tag)DNA (e.g. genomic DNA) or both DNA and RNA.

In embodiments in which DNA is captured, the DNA may be any DNA moleculewhich may occur in a cell. Thus it may be genomic, i.e. nuclear, DNA,mitochondrial DNA or plastid DNA, e.g. chloroplast DNA. In a preferredembodiment, the DNA is genomic DNA.

In embodiments in which RNA is captured, the RNA may be any RNA moleculewhich may occur in a cell. Thus it may be mRNA, tRNA, rRNA, viral RNA,small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), microRNA(miRNA), small interfering RNA (siRNA), piwi-interacting RNA (piRNA),ribozymal RNA, antisense RNA or non-coding RNA. Preferably however it ismRNA.

In the context of RNA, step (f) may be viewed as generating cDNA(particularly tagged cDNA) from the captured RNA, i.e. relating to thesynthesis of the cDNA. This will involve a step of reverse transcription(RT) of the captured RNA, extending the capture probe, which functionsas the RT primer, using the captured RNA as template. Such a stepgenerates so-called first strand cDNA, i.e. an extended probe.

In the context of DNA, step (f) may be viewed as generating DNA(particularly tagged DNA) from the captured DNA, i.e. relating to thesynthesis of a complementary strand of DNA or ligation of one of the DNAstrands to the capture probes. This may involve a step of DNApolymerization, extending the capture probe, which may function as aprimer for the extension, using the captured DNA as template to producea complementary strand of the DNA hybridized to the capture probe.Alternatively, this may involve a step of DNA ligation, extending thecapture probe, which may function as a substrate and optionally thetemplate in a ligation reaction.

As will be described in more detail below, generating a complement ofthe extended probe (e.g. second strand cDNA synthesis) may take place ina separate step, prior to the step of analyzing the extended probes(e.g. the sequence of the extended probes) or may take place as part ofthe analysis step. Thus, for instance, generating a complement of theextended probe (e.g. second strand synthesis) may occur in the firststep of amplification of an extended probe. In some embodiments,generating a complement of the extended probe (e.g. second strandsynthesis) may occur contemporaneously with the extension of the captureprobe (e.g. first strand synthesis) or may be performed immediatelyfollowing the extension of the capture probe (e.g. first strandsynthesis reaction). For instance, second strand synthesis may occurcontemporaneously with the first strand synthesis reaction when atemplate switching reaction is used for second strand synthesis.Template switching reactions are described in detail below.

Thus, in some embodiments, (i.e. when the method is used to captureRNA), the extension reaction comprises the use of a reversetranscriptase enzyme. The desired reverse transcriptase activity may beprovided by one or more distinct reverse transcriptase enzymes, whereinsuitable examples are: M-MLV, MuLV, AMV, HIV, ArrayScript™,MultiScribe™, ThermoScript™, and SuperScript® I, II, and III enzymes. Asused herein, the term “reverse transcriptase” includes not onlynaturally occurring enzymes but also all such modified derivatives,including also derivatives of naturally occurring reverse transcriptaseenzymes.

Particularly preferred reverse transcriptase enzymes for use in themethods of the present application include M-MLV, MuLV, AMV and HIVreverse transcriptase enzymes and derivatives, e.g. sequence-modifiedderivatives, or mutants thereof.

Sequence-modified derivatives or mutants of M-MLV, MuLV, AMV and HIVreverse transcriptase enzymes include mutants that retain at least someof the functional, e.g. reverse transcriptase, activity of the wild-typesequence. Mutations may affect the activity profile of the enzymes, e.g.enhance or reduce the rate of polymerisation, under different reactionconditions, e.g. temperature, template concentration, primerconcentration etc. Mutations or sequence-modifications may also affectthe RNase activity and/or thermostability of the enzyme. The reversetranscriptase enzyme may be provided as part of a composition whichcomprises other components, e.g. stabilizing components, that enhance orimprove the activity of the reverse transcriptase enzyme, such as RNaseinhibitor(s), inhibitors of DNA-dependent DNA synthesis, e.g.actinomycin D. Many sequence-modified derivative or mutants of reversetranscriptase enzymes, e.g. M-MLV, and compositions comprisingunmodified and modified enzymes are known in the art and arecommercially available, e.g. ArrayScript™, MultiScribe™, ThermoScript™,and SuperScript® I, II, III and IV enzymes, and all such enzymes areconsidered to be useful in the methods of the invention.

In some embodiments, (i.e. when the method is used to capture DNA), theextension reaction comprises the use of a DNA polymerase enzyme. Thedesired DNA polymerase activity may be provided by one or more distinctDNA polymerase enzymes. In some embodiments, the DNA polymerase enzymeis from a bacterium, i.e. the DNA polymerase enzyme is a bacterial DNApolymerase enzyme. For instance, the DNA polymerase may be from abacterium of the genus Escherichia, Bacillus, Thermophilus orPyrococcus.

Suitable examples of DNA polymerases that may find utility in themethods of the invention include: E.coli DNA polymerase I, Bsu DNApolymerase, Bst DNA polymerase, Taq DNA polymerase, Klenow fragment, PwoDNA polymerase, Pfu DNA polymerase, T4 DNA polymerase and T7 DNApolymerase enzymes. As used herein, the term “DNA polymerase” includesnot only naturally occurring enzymes but also all such modifiedderivatives, including also derivatives of naturally occurring DNApolymerase enzymes. For instance, in some embodiments, the DNApolymerase may have been modified to remove 5′-3′ exonuclease activity.

Particularly preferred DNA polymerase enzymes for use in the methods ofthe present application include E.coli DNA polymerase I, Bsu DNApolymerase and Klenow fragment enzymes and derivatives, e.g.sequence-modified derivatives, or mutants thereof.

Sequence-modified derivatives or mutants of DNA polymerase enzymesinclude mutants that retain at least some of the functional, e.g.reverse transcriptase, activity of the wild-type sequence. Mutations mayaffect the activity profile of the enzymes, e.g. enhance or reduce therate of polymerisation, under different reaction conditions, e.g.temperature, template concentration, primer concentration etc. Mutationsor sequence-modifications may also affect the exonuclease activityand/or thermostability of the enzyme.

In some embodiments, (i.e. when the method is used to capture DNA), theextension reaction comprises the use of a DNA ligase enzyme. The desiredDNA ligase activity may be provided by one or more distinct DNA ligase.In some embodiments, the DNA ligase enzyme is from a bacterium, i.e. theDNA ligase enzyme is a bacterial DNA ligase enzyme. For instance, theDNA ligase may be T4 DNA ligase. Other enzymes appropriate for theligation step are known in the art and include, e.g. Tth DNA ligase, TaqDNA ligase, Thermococcus sp. (strain 9oN) DNA ligase (9oNTM DNA ligase,New England Biolabs), and AmpligaseTM (Epicentre Biotechnologies).Derivatives, e.g. sequence-modified derivatives, or mutants thereof(defined above) may also find utility in the methods of the invention.

As mentioned above, WO 2018/091676 (herein incorporated by reference),discloses a method which combines step of releasing the probes fromsurface of the solid substrate, e.g. array, with the step of extendingthe probes using the captured nucleic acids as templates for extension.Thus, it is contemplated that step (f) of the method of the presentinvention may be combined with a step of releasing the extended probesfrom the solid substrate.

In embodiments in which the extension and release steps are combined thecapture probes are not restricted to a particular orientation on thearray. In this respect, the combination of the release and extensionsteps eliminates the requirement for a particular orientation of thecapture probes on the solid substrate. However, in some embodiments, itis preferred that the capture probes are immobilized on the solidsubstrate such that they have a free 3′ end capable of functioning as anextension primer.

Thus, in preferred embodiments, the capture probes are immobilized onthe array (preferably directly) via their 5′ end and comprise a nucleicacid molecule with 5′ to 3′:

-   (i) a positional domain that corresponds to the position of the    capture probe on the array, and-   (ii) a capture domain.

Furthermore, as the capture probes may be oriented on the solidsubstrate such that the capture domain is not free or available tointeract with (i.e. bind or hybridise to) the nucleic acid molecules inthe biological specimen (i.e. the capture probes may be immobilized viatheir 3′ ends), step (d) may occur simultaneously with step (e), i.e.step (e) may be performed under conditions that allow (i.e. are suitablefor or facilitate) the nucleic acids of the biological specimen tohybridise to the capture domain in said capture probes. However, inpreferred embodiments (e.g. where the capture probes are immobilized onthe solid substrate such that they have a free 3′ end capable offunctioning as an extension primer, e.g. via their 5′ ends) step (e)(and optionally steps (b), (c) and/or (d)) may be performed underconditions that allow the nucleic acids of the biological specimen tohybridise to the capture domain in said capture probes.

Thus, in embodiments where step (f) of the method of the presentinvention is combined with a step of releasing the capture (e.g.extended) probes from the solid substrate and where the capture probesare immobilized on the solid substrate such that they have a free 3′ endcapable of functioning as an extension primer (e.g. by their 5′ end),some capture probes may be released from the solid substrate prior totheir extension, i.e. some capture probes are released and subsequentlyextended. Moreover, some capture probes may be extended at the same timeas they are released from the solid substrate, i.e. some capture probesare extended and released from the solid substrate simultaneously.

The step of releasing the capture probes (e.g. extended probes) from thesurface of the solid substrate may be achieved in a number of ways. Theprimary aim of the release step is to yield molecules into which thepositional domain of the capture probe (or its complement) isincorporated (or included), such that the DNA, e.g. cDNA molecules ortheir amplicons are “tagged” according to their feature (or position) onthe array. The release step thus untethers or removes DNA, e.g. cDNAmolecules (extended probes) or amplicons thereof from the solidsubstrate (array). The DNA, e.g. cDNA molecules (extended probes) oramplicons include the positional domain or its complement (by virtue ofit being part of the extended probe, e.g. the first strand DNA byextension of the capture probe, and optionally copied in thecomplementary stand of the extended probe (i.e. second strand DNA) ifcomplementary/second strand synthesis takes place on the array, orcopied into amplicons if amplification takes place on the array). Hence,in order to yield sequence analysis data that can be correlated with thevarious regions in the tissue sample it is essential that the extendedprobes (e.g. released extended probes or their complements) comprise thepositional domain of the capture probe (or its complement).

EXAMPLES Example 1

While investigating the utility of transposase-mediated fragmentation inmethods of capture and spatial tagging of DNA from a biological sampleusing methods described in WO 2012/140224, it was determined thatpermeabilization conditions that are typically used in tagmentationreactions in cellular suspensions (e.g., as described (Corces, et. al,2016, supra) are not suitable for biological samples (e.g., tissuesections) immobilized on a substrate, such as an array.

Using the workflow set out in FIG. 27 , the effects of detergents in thepre-permeabilization step were compared. In brief, tissue sections fromfrozen tissue samples were crosslinked in 1% or 4% formaldehyde solutionfor 10 minutes at 25° C. and formaldehyde was quenched by adding 0.125 MGlycine and incubation for 5 minutes at 25° C. The tissue sections wererinsed in DPBS to remove crosslinking reagents. The tissue sections weresubsequently dehydrated with isopropanol and air-dried. These tissuesections are suitable for histological analysis. The tissue sectionswere then re-hydrated in D-PBS prior to pre-permeabilization.

Pre-permeabilization involved incubating the re-hydrated tissue sectionsin: detergents, 0.1% Triton-X-100, IGEPAL 0.1% or Tween 0.1%, Digitonin0.01% and NP-40 0.1% for 10 minutes at 25° C.

The Tn5 transposome was assembled as described in Picelli et al. 2014(supra) and tagmentation was performed using conditions similar to thosein Corces, M.R., et. al., An improved ATAC-seq protocol reducesbackground and enables interrogation of frozen tissues, Nat Methods,vol. 14(10): 959-962 (2017). In particular, the pre-permeabilizationsolution was removed from the tissue sections and 50 µl of tagmentationmix was added to the tissue sections (tagmentation mix):

2x TD buffer Digitonin 1% Tween-20 10% DPBS H₂O Tn5 (MEDS-40 µM) 2X TDbuffer: 25 µl 0.5 µl 0.5 µl 16.5 µl 6.25 µl 1.25 µl. Stock Volume for100 ml Final conc. 1 M Tris HCl pH7.6 1 M MgCl₂ Dimethyl FormamideSterile H₂O 2 ml 1 ml (DMF) 20 ml Up to 100 ml 20 mM 10 mM 20% NA The TDbuffer was adjusted to pH 7.6 with acetic acid prior to the addition ofDMF.

The tissue sections were incubated in the tagmentation mix for 30minutes at 37° C. while shaking at 300 rpm (an adhesive lid was providedto prevent loss of the tagmentation mix). Nucleic acid samples obtainedfrom the tissue samples were analysed for fragment size distribution,e.g. with an Agilent Bioanalyzer (Agilent).

FIG. 30 shows that none of the tested detergents yield the nucleosomalpattern typically associated with tagmentation. While not wishing to bebound by theory, it is hypothesized that none of the detergents used aresufficient for efficient nuclear accessibility for the Tn5 transposasein immobilized tissue sections given the large fragment sizedistribution.

In order to make the nuclear envelope accessible to enzymes insubsequent reactions, tissue sections were then subjected to variouschemical or enzymatic pre-permeabilization conditions.

FIG. 32 shows that successful pre-permeabilization may be obtained usingpepsin in 0.5 M acetic acid or Exonuclease-1 buffer. It was found thatthe acidity of the buffers required for pepsin digestion can inducegenomic fragmentation (FIGS. 31A-C).

The most efficient genomic accessibility achieved, while preservingnuclear integrity, was obtained using collagenase in the presence ofBSA. The time of digestion at 37° C. can be adjusted according to thenature of the tissue. For example, mouse brain tissues can bepre-permeabilized for 20 minutes in collagenase solution for optimalaccessibility (FIG. 32C). Longer permeabilization incubation times incollagenase, Pepsin, or Proteinase-K (FIG. 31D) can be used to capturegenomic DNA fragments regardless of their chromatin accessibility.

Example 2 Transposome Assembly

Tn5 transposase may be produced as previously described (Picelli et al.,2014 supra). In brief, Tn5 transposase protein monomers are produced andpurified and subsequently loaded with the oligonucleotides of interest.The ssDNA oligonucleotides contain mosaic ends for Tn5 recognition andare annealed to form a dsDNA mosaic end oligonucleotide (MEDS) that isrecognized by Tn5 during dimer assembly. The oligonucleotides maycontain desired 5′ overhangs for functionalization of tagmented DNA. Theoligonucleotide can also contain an additional single stranded domains.

Effects of 5′ Phosphorylation on Tagmentation

As described above, the functional domain of the MEDS can employ aphosphorylated 5′ end to allow ligation of tagmented DNA to the captureprobes. This can be achieved by assembling Tn5 with 5′ phosphorylatedMEDS oligonucleotides in solution.

It was found that tagmentation using in-solution assembly of 5′phosphorylated MEDS onto Tn5 protein is inefficient (FIG. 33C). Asunphosphorylated MEDS oligonucleotides with 5′ overhangs have accessible5′ hydroxyl groups outside of the mosaic-end Tn5 binding site, theassembled complexes were phosphorylated by exposing these 5′ ends of theMEDS-Tn5 complexes to T4-polynucleotide kinase (T4-PNK) in the presenceof ATP. Specifically, 2.5µl of the Tn5 assembled complex was added to areaction mixture containing:

T4 PNK Reaction Buffer (10x) 1 µl ATP (10 mM) 1 µl T4 PNK (10 U/µl) 0.5µl Nuclease free H₂O 5 µl

The reaction was carried out at 37° C. for 30 minutes and thephosphorylated Tn5 complex was termed “Phospho-Tn5”.

Tagmentation was performed as described in Example 1, wherein in thereaction mixture containing the “Phospho-Tn5” (PNK-MEDS-Tn5) contained:

2x TD buffer 25 µl Digitonin 1% 0.5 µl Tween-20 10% 0.5 µl DPBS 16.5 µlH₂O 2.5 µl “Phospho-Tn5” 5 µl

It was found that the phosphorylated MEDS-Tn5 complexes (PNK-MEDS-Tn5)retain most of the transposition activity unlike MEDS-Tn5 assembliesgenerated in-solution in the presence of excess MEDS (FIGS. 34 and 35 ).

Example 3

Capturing of the tagments onto the substrate, spatially barcoded array,can be performed using two main capture strategies, hybridization andligation. The strategy may depend on the purpose of the experiment,e.g., whether the tagments are to be captured alone or simultaneouslywith mRNA-transcripts. Representative embodiments for each capturestrategy are described below.

Simultaneous Capture of Tagments and mRNAs Using Hybridization

Simultaneous capture of tagments and mRNA on standard spatially barcodedarrays is performed using hybridization of poly(A) tails of mRNAtranscripts and poly(A) tailed tagmented DNA to the polyT sequences onthe capture probes (See e.g., WO 2012/140224). This is possible byadding a poly(A) tail to the tagments, e.g. by gap-filling and ligatingbreaks in the tagmented DNA and subsequently adding a poly(A) tail witha terminal transferase enzyme, such as terminal transferasedeoxynucleotidyl transferase. This will create tagments with 3′-poly(A)sticky ends, mimicking the poly(A) tail of mRNA, thus allowing forsimultaneous capture of the tagmented DNA and the mRNA transcripts (FIG.37 ). Optimally, the length of the obtained sticky-end of poly-A shouldbe 18 bases or longer. Alternatively, instead of a sequential reaction(e.g., gap filling followed by a terminal transferase), a singlereaction with a polymerase (e.g., DNA polymerase) may be performed. Thepost-hybridization steps are identical as described in Ståhl P.L., etal. Visualization and analysis of gene expression in tissue sections byspatial transcriptomics Science, vol. 353, 6294, pp. 78-82 (2016)).

Capturing Tagments Using Ligation

Following tagmentation with Tn5, a further permeabilization step isperformed to allow the intra-nuclear tagments to diffuse out of thetissue section and ligate to the surface probes onto the array. Theligation uses a partially double stranded capture probe, comprising acapture domain oligonucleotide (e.g., a splint oligonucleotide) and asurface probe. The capture domain oligonucleotide may be viewed as a“splint oligonucleotide” that hybridizes to the adapter sequence (SEQ IDNO. 18) ligated to the tagmented DNA by the hyperactive Tn5 transposaseand a complementary sequence on a surface probe (FIG. 38 ). The ligationincubation mix contains 1x T4 DNA ligase buffer, 0.02 µM splintoligonucleotide, 0.01 µM BSA, nuclease-free water and T4 DNA ligase at avolume half of the T4 DNA ligase buffer. This mix is added to each ofthe array-wells and incubated at room temperature overnight.

Example 4

Ligation of purified DNA tagments from a whole human genome to a captureprobe on the substrate surface (e.g., a partially double strandedcapture probe comprising a surface probe and a splint oligonucleotidewith a capture domain) was performed, followed by qPCR and bioanalyzeranalysis (FIG. 39 ).

Immobilization of the surface probe portion of the capture probe (IDT)to enable ligation was performed on the surface of Codelink Activatedmicroscope glass slides (#DN01-0025, Surmodics), according to themanufacturer’s instructions. The oligonucleotide (e.g., surface probe)immobilized on the surface is shown below (SEQ ID NO. 15):

[AmC6]UUUUUGACTCGTAATACGACTCACTATAGGGACACGACGCTCTTCCGATCTNNNNNNNNTGCACGCGGTGTACAGACGT

Hybridization of splint oligonucleotides (2 µM diluted in PBS) tosurface probes was performed for 30 min at 44° C. (FIG. 40 ) thusgenerating the capture probe.

Ligation and Strand Displacement Hybridization

Ligation was performed for 2 hours at 37° C. (0.005 weiss U/µl T4 DNAligase, 0.2 mg/ml BSA, 1x T4 DNA Ligase Buffer, 8.75 ng/µl tagments) byadding 70 µl to each well (FIG. 41A). After ligation, stranddisplacement polymerization was performed (0.27 U/µl DNA polymerase I(#18010-017, Invitrogen), 0.27 µg/µl BSA, 0.6 mM dNTPs, 1x DNA pol 1Reaction Buffer) by incubation at 37° C. for 1 hour.

Release of Capture Probes and Downstream Analysis

For each well, 70 µl release mix (0.20 µg/µl BSA, 0.1U/µl USER Enzyme(#M5505, NEB) was added and incubated at 37° C. for 1 hour and 65 µlfrom each well were collected. Volume reduction using a SpeedVac down to~10 µl was performed. A qPCR reaction was then performed containing atotal reaction volume of 10 µl (1xKAPA HiFi HotStart ReadyMix (#KK2601,KAPA Biosystems), 1x EVA green (#31000, Biotium), and primers (25 µM)).Amplification was performed with the following protocol: 72° C. for 10minutes, 98° C. for 3 minutes, followed by cycling at 98° C. for 20seconds, 60° C. for 30 seconds and 72° C. for 30 seconds. Two primerpairs were used for qPCR, one pair that included A-short forward andNextera reverse (covers ligated part + capture probe; SEQ ID NOs. 21 and20, respectively) and the other pair that included Nextera forward andNextera reverse (covers the tagment-part only). The second pair (Nexteraforward and Nextera reverse; SEQ ID NOs. 16 and 20, respectively) servedas a control for the ligation since only hybridization of the tagment tothe splint oligonucleotide is required, and not ligation (FIG. 40 ).

The samples were purified as described elsewhere (Lundin et al.,Increased Throughput by Parallelization of Library Preparation forMassive Sequencing, PLOS ONE, 5(4), doi.org/10.1371/journal.pone.0010029(2010) which is herein incorporated by reference) and then diluted in 20µl elution buffer (#19086, Qiagen). Average fragment length wasdetermined using the DNA HS Kit (Agilent) with a 2100 Bioanalyzeraccording to the manufacturer’s protocol (FIGS. 41B-C). These resultsshow successful capture of fragments from the DNA not restricted to openchromatin and that the negative controls (at two levels) were truenegatives.

Example 5

Ligation of purified DNA tagments from an immobilized tissue sections tocapture probes on a substrate surface was performed, followed by qPCRand bioanalyzer analysis according to the workflow shown in FIG. 42 .Capture probe (e.g., the surface probe of the capture probe)immobilization and hybridization of the splint oligonucleotide wereperformed as described in Example 4. Tissue handling and additionalpermeabilization optimization conditions are described in this Example.

Fixation, Permeabilization and DNA Tagmentation

Tissue sections (10 µm) were placed onto the arrays and incubated at 37°C. for 1 minute followed by crosslinking in 4% formaldehyde solution for10 minutes at 25° C. The tissue sections were then rinsed in PBS toremove crosslinking reagents. Pre-permeabilization was performed usingcollagenase in HBSS buffer (0.2 U/µl collagenase, 0.2 mg/µl BSA) at 37°C. for 20 min.

Pre-permeabilization using either Proteinase K (#19131, Qiagen) and PKDBuffer (#1034963, Qiagen), at a ratio of 1:8 at 37° C. for 10 minutes or15% trypsin at 37° C. for 10 minutes was performed. The procedure wasperformed according to the workflow shown in FIG. 42 including qPCR andbioanalyzer analysis and the results are shown in FIG. 43 ,

These results show that tagmentation, ligation, and downstream analysis(e.g., qPCR and bioanalyzer analysis) can be performed on immobilized(e.g., fixed) biological samples (e.g., tissue section).

Example 6

Ligation of purified DNA tagments (via adapters) from immobilized tissuesections to surface probes on a substrate surface was performed followedby qPCR and hybridization of Cy5-labeled oligonucleotides. The workflowfollows Example 5, but with the following changes:

Pre-permeabilization and permeabilization were performed using onlyProteinase K (#19131, Qiagen) and PKD Buffer (#1034963, Qiagen), at aratio of 1:8 at 37° C. for 10 minutes and tagmentation time was extendedto 45 minutes instead of 30 minutes.

Additionally, a parallel downstream analysis after tissue removalincluded surface-based denaturation (1M NaOH) of ligated tagments atroom temperature for 10 minutes followed by hybridization of Cy5-labeledoligonucleotides. FIG. 45 shows qPCR data from two experiments (FIGS.45A-B) with a negative control (unphosphorylated tagments) (FIG. 45C).FIG. 45D shows an image of the spatial capture pattern of the DNAtagments with Cy5-labeled oligonucleotides. The bioanalyzer results gavea reduced signal in tissue section 2 (FIG. 45B), and a more sporadicpeak pattern. FIG. 45D shows that the Cy5-image (right) resembles thehematoxylin-eosin image (left).

SEQ ID NO: 1 - Tn5 Transposase

MITSALHRAADWAKSVFSSAALGDPRRTARLVNVAAQLAKYSGKSITISSEGSEAMQEGAYRFIRNPNVSAEAIRKAGAMQTVKLAQEFPELLAIEDTTSLSYRHQVAEELGKLGSIQDKSRGWWVHSVLLLEATTFRTVGLLHQEWWMRPDDPADADEKESGKWLAAAATSRLRMGSMMSNVIAVCDREADIHAYLQDKLAHNERFVVRSKHPRKDVESGLYLYDHLKNQPELGGYQISIPQKGVVDKRGKRKNRPARKASLSLRSGRITLKQGNITLNAVLAEEINPPKGETPLKWLLLTSEPVESLAQALRVIDIYTHRWRIEEFHKAWKTGAGAERQRMEEPDNLERMVSILSFVAVRLLQLRESFTLPQALRAQGLLKEAEHVESQSAETVLTPDECQLLGYLDKGKRKRKEKAGSLQWAYMAIARLGGFMDSKRTGIASWGALWEGWEALQSKLDGFLAAKDLMAQGIKI

SEQ ID NO: 2 - Bacteriophage Mu Transposase

MKEWYTAKELLGLAGLPKQATNITRKAQREGWEFRQVAGTKGVSFEFNIKSFPVALRAEILLQQGRIETSQGYFEIARPTLEAHDYDREALWSKWDNASDSQRRLAEKWLPAVQAADEMLNQGISTKTAFATVAGHYQVSASTLRDKYYQVQKFAKPDWAAALVDGRGASRRNVHKSEFDEDAWQFLIADYLRPEKPAFRKCYERLELAAREHGWSIPSRATAFRRIQQLDEAMVVACREGEHALMHLIPAQQRTVEHLDAMQWINGDGYLHNVFVRWFNGDVIRPKTWFWQDVKTRKILGWRCDVSENIDSIRLSFMDVVTRYGIPEDFHITIDNTRGAANKWLTGGAPNRYRFKVKEDDPKGLFLLMGAKMHWTSVVAGKGWGQAKPVERAFGVGGLEEYVDKHPALAGAYTGPNPQAKPDNYGDRAVDAELFLKTLAEGVAMFNARTGRETEMCGGKLSFDDVFEREYARTIVRKPTEEQKRMLLLPAEAVNVSRKGEFALKVGGSLKGAKNVYYNMALMNAGVKKVVVRFDPQQLHSTVYCYTLDGRFICEAECLAPVAFNDAAAGREYRRRQKQLKSATKAAIKAQKQMDALEVAELLPQIAEPEAPESRIVGIFRPSGNTERVKNQERDDEYETERDEYLNHSL DILEQNRRKKAI

SEQ ID NO: 3 - Pepsin

IGDEPLENYLDTEYFGTIGIGTPAQDFTVIFDTGSSNLWVPSVYCSSLACSDHNQFNPDDSSTFEATSQELSITYGTGSMTGILGYDTVQVGGISDTNQIFGLSETEPGSFLYYAPFDGILGLAYPSISASGATPVFDNLWDQGLVSQDLFSVYLSSNDDSGSVVLLGGIDSSYYTGSLNWVPVSVEGYWQITLDSITMDGETIACSGGCQAIVDTGTSLLTGPTSAIANIQSDIGASENSDGEMVISCSSIDSLPDIVFTINGVQYPLSPSAYILQDDDSCTSGFEGMDVPTSSGELWILGDVFIRQYYTVFDRANNKVGLAPVA

SEQ ID NO: 4 - Pepsin

AATLVSEQPLQNYLDTEYFGTIGIGTPAQDFTVIFDTGSSNLWVPSIYCSSEACTNHNRFNPQDSSTYEATSETLSITYGTGSMTGILGYDTVQVGGISDTNQIFGLSETEPGSFLYYAPFDGILGLAYPSISSSGATPVFDNIWDQGLVSQDLFSVYLSSNEESGSVVIFGDIDSSYYSGSLNWVPVSVEGYWQITVDSITMNGESIACSDGCQAIVDTGTSLLAGPTTAISNIQSYIGASEDSSGEVVISCSSIDSLPDIVFTINGVQYPVPPSAYILQSNGICSSGFEGMDISTSSGDLWILGDVFIRQYFTVFDRGNNQIGLAPVA

SEQ ID NO: 5 - Collagenase

IANTNSEKYDFEYLNGLSYTELTNLIKNIKWNQINGLFNYSTGSQKFFGDKNRVQAIINALQESGRTYTANDMKGIETFTEVLRAGFYLGYYNDGLSYLNDRNFQDKCIPAMIAIQKNPNFKLGTAVQDEVITSLGKLIGNASANAEVVNNCVPVLKQFRENLNQYAPDYVKGTAVNELIKGIEFDFSGAAYEKDVKTMPWYGKIDPFINELKALGLYGNITSATEWASDVGIYYLSKFGLYSTNRNDIVQSLEKAVDMYKYGKIAFVAMERITWDYDGIGSNGKKVDHDKFLDDAEKHYLPKTYTFDNGTFIIRAGDKVSEEKIKRLYWASREVKSQFHRVVGNDKALEVGNADDVLTMKIFNSPEEYKFNTNINGVSTDNGGLYIEPRGTFYTYERTPQQSIFSLEELFRHEYTHYLQARYLVDGLWGQGPFYEKNRLTWFDEGTAEFFAGSTRTSGVLPRKSILGYLAKDKVDHRYSLKKTLNSGYDDSDWMFYNYGFAVAHYLYEKDMPTFIKMNKAILNTDVKSYDEIIKKLSDDANKNTEYQNHIQELADKYQGAGIPLVSDDYLKDHGYKKASEVYSEISKAASLTNTSVTAEKSQYFNTFTLRGTYTGETSKGEFKDWDEMSKKLDGTLESLAKNSWSGYKTLTAYFTNYRVTSDNKVQYDVVFHGVLTDNADISNNKAPIAKVTGPSTGAVGRNIEFSGKDSKDEDGKIVSYDWDFGDGATSRGKNSVHAYKKAGTYNVTLKVTDDKGATATESFTIEIKNEDTTTPITKEMEPNDDIKEANGPIVEGVTVKGDLNGSDDADTFYFDVKEDGDVTIELPYSGSSNFTWLVYKEGDDQNHIASGIDKNNSKVGTFKSTKGRHYVFIYKHDSASNISYSLNIKGLGNEKLKEKENNDSSDKATVIPNFNTTMQGSLLGDDSRDYYSFEVKEEGEVNIELDKKDEFGVTWTLHPESNINDRITYGQVDGNKVSNKVKLRPGKYYLLVYKYSGSG NYELRVNK

SEQ ID NO: 6 - Collagenase

VQNESKRYTVSYLKTLNYYDLVDLLVKTEIENLPDLFQYSSDAKEFYGNKTRMSFIMDEIGRRAPQYTEIDHKGIPTLVEVVRAGFYLGFHNKELNEINKRSFKERVIPSILAIQKNPNFKLGTEVQDKIVSATGLLAGNETAPPEVVNNFTPILQDCIKNIDRYALDDLKSKALFNVLAAPTYDITEYLRATKEKPENTPWYGKIDGFINELKKLALYGKINDNNSWIIDNGIYHIAPLGKLHSNNKIGIETLTEVMKVYPYLSMQHLQSADQIKRHYDSKDAEGNKIPLDKFKKEGKEKYCPKTYTFDDGKVIIKAGARVEEEKVKRLYWASKEVNSQFFRVYGIDKPLEEGNPDDILTMVIYNSPEEYKLNSVLYGYDTNNGGMYIEPEGTFFTYEREAQESTYTLEELFRHEYTHYLQGRYAVPGQWGRTKLYDNDRLTWYEEGGAELFAGSTRTSGILPRKSIVSNIHNTTRNNRYKLSDTVHSKYGASFEFYNYACMFMDYMYNKDMGILNKLNDLAKNNDVDGYDNYIRDLSSNYALNDKYQDHMQERIDNYENLTVPFVADDYLVRHAYKNPNEIYSEISEVAKLKDAKSEVKKSQYFSTFTLRGSYTGGASKGKLEDQKAMNKFIDDSLKKLDTYSWSGYKTLTAYFTNYKVDSSNRVTYDVVFHGYLPNEGDSKNSLPYGKINGTYKGTEKEKIKFSSEGSFDPDGKIVSYEWDFGDGNKSNEENPEHSYDKVGTYTVKLKVTDDKGESSVSTTTAEIKDLSENKLPVIYMHVPKSGALNQKVVFYGKGTYDPDGSIAGYQWDFGDGSDFSSEQNPSHVYTKKGEYTVTLRVMDSSGQMSEKTMKIKITDPVYPIGTEKEPNNSKETASGPIVPGIPVSGTIENTSDQDYFYFDVITPGEVKIDINKLGYGGATWVVYDENNNAVSYATDDGQNLSGKFKADKPGRYYIHLYMFNGSYMPYRINIEGSVGR

SEQ ID NO: 7 - Proteinase K

AAQTNAPWGLARISSTSPGTSTYYYDESAGQGSCVYVIDTGIEASHPEFEGRAQMVKTYYYSSRDGNGHGTHCAGTVGSRTYGVAKKTQLFGVKVLDDNGSGQYSTIIAGMDFVASDKNNRNCPKGVVASLSLGGGYSSSVNSAAARLQSSGVMVAVAAGNNNADARNYSPASEPSVCTVGASDRYDRRSSFSNYGSVLDIFGPGTSILSTWIGGSTRSISGTSMATPHVAGLAAYLMTLGKTTAASACRYIADTANKGDLSNIPFGTVNLLAYNNYQA

SEQ ID NO: 8 Tn5 Mosaic end sequence

CTGTCTCTTA TACACATCT

SEQ ID NO: 9 - Mu Transposase Recognition Sequence

TGAAGCGGCG CACGAAAAAC GCGAAAG

SEQ ID NO 10 - Mu Transposase Recognition Sequence

GCGTTTCACG ATAAATGCGA AAA

SEQ ID NO: 11 - Mu Transposase Recognition Sequence

CTGTTTCATT TGAAGCGCGA AAG

SEQ ID NO: 12 - Mu Transposase Recognition Sequence

TGTATTGATT CACTTGAAGT ACGAAAA

SEQ ID NO: 13 - Mu Transposase Recognition Sequence

CCTTAATCAA TGAAACGCGA AAG

SEQ ID NO: 14 - Mu Transposase Recognition Sequence

TTGTTTCATT GAAAATACGA AAA

SEQ ID NO: 15 - Surface probe of the capture probe

UUUUUGACTC GTAATACGAC TCACTATAGG GACACGACGC TCTTCCGATC TNNNNNNNNTGCACGCGGTG TACAGACGT

SEQ ID NO: 16 - First adapter

GTCTCGTGGG CTCGG

SEQ ID NO: 17 - Capture domain

CCGAGCCCAC GAGAC

SEQ ID NO: 18 - Hybridization domain

TGCACGCGGT GTACAGACGT

SEQ ID NO: 19 - Splint oligonucleotide complementary to hybridizationdomain

ACGTCTGTAC ACCGCGTGCA

SEQ ID NO: 20 - Second adapter

TCGTCGGCAG CGTC

SEQ ID NO: 21 - A-short forward

ACACGACGCT CTTCCGATCT

What is claimed is:
 1. A method for determining a spatial location ofaccessible DNA in a tissue sample, the method comprising: (a) providingan array comprising a plurality of capture probes affixed thereon,wherein a capture probe of the plurality of capture probes comprises (i)a spatial barcode and (ii) a capture domain; (b) permeabilizing thetissue sample; (c) contacting a transposome with the permeabilizedtissue sample to insert transposon end sequences into the accessibleDNA, thereby generating a fragmented nucleic acid sequence, and addingto an end of the fragmented nucleic acid sequence a first adaptercomprising a sequence substantially complementary to a sequence of thecapture domain; (d) hybridizing the first adapter of the fragmentednucleic acid sequence to the capture domain of the capture probe; and(e) determining the spatial barcode sequence or a complement thereof,and the fragmented nucleic acid sequence adjacent to the first adapterand the transposon end sequence, or a complement thereof, therebydetermining the spatial location of accessible DNA in the tissue sample.2. The method of claim 1, wherein adding the first adapter to the end ofthe fragmented nucleic acid sequence in step (c) comprises ligating thefirst adapter to a 5′ end of the fragmented nucleic acid sequence andwherein determining the fragmented nucleic acid sequence comprisesdetermining a sequence 3′ to the first adapter and the transposon endsequence.
 3. The method of claim 1, wherein the array comprises one ormore features selected from the group consisting of a bead, an inkjetspot, a masked spot, a well, and a hydrogel pad.
 4. The method of claim1, wherein the capture probe further comprises a cleavage domain, afunctional domain, a unique molecular identifier, or combinationsthereof.
 5. The method of claim 1, wherein the transposome comprises atransposase enzyme and transposon ends for inserting into accessible DNAand wherein the transposon ends further comprise a first adapter and asecond adapter and the 5′ end of the first adapter is complexed with afirst monomer of the transposase enzyme and a 5′ end of the secondadapter complexed with a second monomer or the transposase enzyme, andwherein the 5′ ends of the adapters are phosphorylated.
 6. The method ofclaim 1, wherein the capture probe comprises (i) a hybridization domain,and (ii) a splint oligonucleotide hybridized to the hybridizationdomain, or a portion thereof, and wherein the splint oligonucleotidecomprises a capture domain, and wherein the splint oligonucleotidehybridizes to the first adapter or a portion thereof, of the fragmentednucleic acid sequence.
 7. The method of claim 6, wherein the methodfurther comprises ligating the first adapter of the fragmented nucleicacid sequence to the surface probe using the splint oligonucleotide as atemplate, wherein the ligating is performed with a DNA ligase.
 8. Themethod of claim 1, wherein step (d) comprises extending a 3′ end of thecapture probe using the hybridized fragmented nucleic acid sequence as atemplate, wherein the extending step is performed using a DNA polymerasehaving strand displacement activity.
 9. The method of claim 1, whereinthe method further comprises performing gap repair of single-strandedbreaks in the fragmented nucleic acid sequence, wherein the gap repairis performed after adding the first adapter to the end of the fragmentednucleic acid sequence.
 10. The method of claim 1, wherein thetransposome comprises a Tn5 transposase enzyme, a Mu transposase enzyme,a Tn7 transposase enzyme, or functional derivatives thereof, and whereinthe Tn5 transposase enzyme comprises a sequence that is at least 80%identical to SEQ ID NO: 1 and wherein the transposon end sequencecomprises a sequence that is at least 80% identical to SEQ ID NO:
 8. 11.The method of claim 1, wherein permeabilizing the tissue samplecomprises an enzymatic permeabilization condition comprising aproteinase K enzyme, a proteinase K-like enzyme, or a functionalequivalent thereof comprising a sequence that is at least 80% identicalto SEQ ID NO:
 7. 12. The method of claim 1, wherein the determining instep (e) comprises sequencing (i) the spatial barcode or the complementthereof, and (ii) the fragmented nucleic acid sequence adjacent to thefirst adapter and the transposon end sequence or a complement thereof.13. The method of claim 1, wherein the method further comprises imagingthe tissue sample before and/or after contacting the tissue sample withthe array.
 14. The method of claim 1, wherein the tissue sample isdisposed on the array comprising a plurality of capture probes.
 15. Themethod of claim 14, wherein a diffusion-resistant medium is applied tothe tissue sample, such that the tissue sample is disposed between thearray and the diffusion-resistant medium.
 16. The method of claim 1,wherein the tissue sample is disposed on a substrate.
 17. The method ofclaim 16, wherein the method further comprises aligning the substratewith the array, such that at least a portion of the tissue sample isaligned with at least a portion of the array.
 18. The method of claim17, further comprising migrating the fragmented nucleic acid sequence tothe array.
 19. The method of claim 17, further comprising anelectrophoretic transfer system wherein the tissue sample is disposedbetween a cathode on the array and wherein the array is disposed betweenthe tissue sample and an anode.
 20. The method of claim 19, wherein anelectric field is applied to the electrophoretic transfer system,thereby migrating the fragmented nucleic acid sequence to the anode. 21.The method of claim 1, further comprising at step (d) hybridizing mRNAfrom the tissue sample to a second capture domain of a second captureprobe on the array, wherein the second capture probe comprises a secondspatial barcode, a cleavage domain, a functional domain, a uniquemolecular identifier, or combinations thereof and extending the secondcapture probe using the hybridized mRNA as a template.
 22. The method ofclaim 21, wherein the capture domain that hybridizes to the firstadaptor and the second capture domain are the same; the capture domainand the second capture domain comprise a poly(T) sequence; or thecapture domain and the second capture domain are different.
 23. Themethod of claim 1, wherein the tissue sample is a tissue section,wherein the tissue section is a fresh-frozen tissue section or a fixedtissue section, wherein the fixed tissue section is a formalin-fixedparaffin-embedded tissue section, a paraformaldehyde-fixed tissuesection, a methanol fixed tissue section, or an acetone fixed tissuesection.
 24. The method of claim 21, further comprising determining (i)the sequence of the second spatial barcode or a complement thereof, and(ii) the mRNA sequence or a complement thereof, thereby determining thespatial location of the mRNA in the tissue sample.
 25. The method ofclaim 24, wherein the spatial location of accessible DNA in the tissuesample is used to generate a genome wide chromatin accessibility mapand/or wherein the spatial location of the mRNA in the tissue sample isused to generate a spatial transcriptomic profile and/or correlating thegenome wide chromatin accessibility map and the spatial transcriptomicprofile to identify changes in gene expression, transcription factorbinding, or tissue sample methylation levels.